LIBRARY 

OF   THE 

UNIVERSITY  OF  CALIFORNIA. 

Class 


ELEMENTS 


OF 


WATER  BACTERIOLOGY 


WITH  SPECIAL  REFERENCE  TO 


SANITARY   WATER   ANALYSIS. 


BY 

SAMUEL  GATE   PRESCOTT, 

Assistant  Professor  of  Industrial  Biology, 
AND 

CHARLES-EDWARD  AMORY  WINSLOW, 

Instructor  in  Sanitary  Bacteriology, 

IN  THE 

MASSACHUSETTS  INSTITUTE  OF  TECHNOLOGY. 


FIRST    EDITION. 
FIRST    THOUSAND 


M  7  V 

NEW  YORK : 

JOHX  WILEY   &   SONS^ 
LONDON  :   CHAPMAN   &   HALL,   LIMITED. 
1904. 


Copyright,  1904, 

BY 
S.  C.  PRESCOTT 

AND 

C.-E.  A.  WINSLOW. 


ROBERT  DRUMMOND,   PRINTER,   NEW  YORK. 


DEDICATED 
TO 

£2Ufllfam  Cijompson 

BY   TWO   OF   HIS   PUPILS, 
AS  A    TOKEN    OF    GRATEFUL    AFFECTION. 


128138 


PREFACE. 


THE  general  awakening  of  the  community  to  the 
importance  of  the  arts  of  sanitation — accelerated  by  the 
rapid  growth  of  cities  and  the  new  problems  of  urban 
life — demands  new  and  accurate  methods  for  the  study 
of  the  microbic  world.  Bacteriology  has  long  since 
ceased  to  be  a  subject  of  interest  and  importance  to 
the  medical  profession  merely,  but  has  become  intimately 
connected  with  the  work  of  the  chemist,  the  biologist, 
and  the  engineer.  To  the  sanitary  engineer  and  the 
public  hygienist  a  knowledge  of  bacteriology  is  indis- 
pensable. 

In  the  swift  development  of  this  science  during  the 
last  ten  years  perhaps  no  branch  of  bacteriology  has 
made  more  notable  progress  than  that  which  relates  to 
the  sanitary  examination  of  water.  After  a  brief  period 
of  extravagant  anticipation,  and  an  equally  unreason- 
able era  of  neglect  and  suspicion,  the  methods  of  the 
practical  water  bacteriologist  have  gradually  made  their 
way,  until  it  is  recognized  that,  on  account  of  their  deli- 
cacy, their  directness,  and  their  certainty,  these  methods 
now  furnish  the  final  criterion  of  the  sanitary  condition 
of  a  potable  water. 


vi  PREFACE. 

A  knowledge  of  the  new  science  early  became  so 
indispensable  for  the  sanitary  expert  that  a  special 
course  in  the  Bacteriology  of  Water  and  Sewage  has  for 
some  years  been  given  to  students  of  biology  and  sani- 
tary engineering  in  the  Biological  Department  of  the 
Massachusetts  Institute  of  Technology.  For  workers  in 
this  course  the  present  volume  has  been  especially 
prepared,  and  it  is  fitting,  we  think,  that  such  a  manual 
should  proceed  from  an  institution  whose  faculty,  gradu- 
ates, and  students  have  had  a  large  share  in  shaping  the 
science  and  art  of  which  it  treats.  We  shall  be  grati- 
fied, however,  if  its  field  of  usefulness  extends  to  those 
following  similar  courses  in  other  institutions,  or  occupied 
professionally  in  sanitary  work. 

The  treatment  of  the  subject  in  the  many  treatises  on 
General  Bacteriology  and  Medical  Bacteriology  is  neither 
special  enough  nor  full  enough  for  modern  needs.  The 
classic  work  of  Grace  and  Percy  Frankland  is  now  ten 
years  old;  and  even  Horrocks'  valuable  " Bacteriological 
Examination  of  Water"  requires  to  be  supplemented  by 
an  account  of  the  developments  in  quantitative  analysis 
which  have  taken  place  on  this  side  of  the  Atlantic. 

It  is  for  us  a  matter  of  pride  that  Water  Bacteriology 
owes  much  of  its  value,  both  in  exactness  of  method 
and  in  common-sense  interpretation,  to  American  sani- 
tarians. The  English  have  contributed  researches  of  the 
greatest  importance  on  the  significance  of  certain  intesti- 
nal bacteria-,  but  with  this  exception  the  best  work 
on  the  bacteriology  of  water  has,  in  our  opinion,  been 
done  in  this  country.  Smith,  Sedgwick,  Fuller,  Whipple, 
Jordan,  and  their  pupils  and  associates  (not  to  mention 


PREFACE.  vii 

others)  have  been  pioneers  in  the  development  of  this 
new  field  in  sanitary  science.  To  gather  the  results  of 
their  work  together  in  such  form  as  to  give  a  correct 
idea  of  the  best  American  practice  is  the  purpose  of 
this  little  book;  and  this  we  have  tried  to  do,  with  such 
completeness  as  shall  render  the  volume  of  value  to  the 
expert  and  at  the  same  time  with  such  freedom  from 
undue  technicality  as  to  make  it  readable  for  the  layman. 
It  should  be  distinctly  understood  that  students  using 
it  are  supposed  to  have  had  beforehand  a  thorough 
course  in  general  bacteriology,  and  to  be  equipped  for 
advanced  work  in  special  lines. 

THE  BIOLOGICAL  LABORATORIES, 

MASS.  INSTITUTE  OF  TECHNOLOGY, 

BOSTON,  March  10,  1904. 


TABLE  OF  CONTENTS. 


CHAPTER   I. 

PAGE 

THE  BACTERIA  IN  NATURAL  WATERS 3 

CHAPTER  H. 
THE  QUANTITATIVE  BACTERIOLOGICAL  EXAMINATION  OF  WATER     19 

CHAPTER  III. 

THE  INTERPRETATION  OF  THE  QUANTITATIVE  BACTERIOLOGICAL 
ANALYSIS 35 

CHAPTER  IV. 

DETERMINATION  OF  THE  NUMBER  OF  ORGANISMS  DEVELOPING 
AT  THE  BODY  TEMPERATURE 43 

CHAPTER  V. 
THE  ISOLATION  OF  SPECIFIC  PATHOGENES  FROM  WATER 49 

CHAPTER  VI. 
METHODS  FOR  THE  ISOLATION  OF  THE  COLON  BACILLUS 58 

CHAPTER  VII. 
SIGNIFICANCE  OF  THE  PRESENCE  OF  B.  COLI  IN  WATER 74 

CHAPTER  VIII. 

PRESUMPTIVE  TESTS  FOR  B.  COLI 94 

ix 


x  TABLE  OF  CONTENTS. 

CHAPTER  IX. 

PAOt 

OTHER  INTESTINAL  BACTERIA 99 

CHAPTER   X. 

THE  SIGNIFICANCE  AND  APPLICABILITY   OF  THE   BACTERIOLOG- 
ICAL EXAMINATION. 109 

APPENDIX 119 

REFERENCES 129 

INDEX 143 


ELEMENTS  OF  WATER  BACTERIOLOGY 


ELEMENTS  OF  WATER  BACTERIOLOGY. 


CHAPTER  I.      . 

THE  BACTERIA  IN  NATURAL  WATERS. 

BACTERIA  are  the  most  numerous  and  the  most  widely 
distributed  of  living  things.  They  are  present  not  merely 
at  the  surface  of  the  earth  or  in  the  bodies  of  water  which 
partially  cover  it,  as  is  the  case  with  most  other  living 
things,  but  in  the  soil  itself,  and  in  the  air  above,  and  in 
the  waters  under  the  earth. 

Probably  no  organisms  are  more  sensitive  to  external 
conditions,  and  none  respond  more  quickly  to  slight 
changes  in  their  environment.  Temperature,  moisture, 
and  oxygen  are  of  importance  in  controlling  their  distri- 
bution; but  the  most  significant  factor  is  generally  the 
amount  of  food  supply.  Bacteria  and  decomposing  or- 
ganic matter  are  always  associated,  and  for  this  reason 
a  brief  consideration  of  the  general  relation  of  bacteria 
to  their  sources  of  food  supply  and  to  other  forms  of 
organic  life  must  precede  the  study  of  their  distribution 
in  any  special  medium. 

3 


4  ELEMENTS  OF  WATER  BACTERIOLOGY. 

First,  then,  the  bacteria,  though  possessing  greater  con- 
structive power  than  any  animal  organism,  lack  the  power 
of  green  plants  to  build  up  their  own  food  from  purely 
inorganic  materials,  and  must  live  upon  the  products  of 
the  growth  of  higher  forms.  A  few  species  which  have 
become  adapted  to  a  parasitic  or  semi-parasitic  mode 
of  life  occur  on  the  surface  of  the  normal  plant  or  animal 
body  or  penetrate  the  deeper  layers  of  diseased  tissues, 
feeding  upon  the 'fluids  of  the  body  or  on  the  extraneous 
material  collected  upon  its  surface.  Even  these  bacteria, 
however,  may  generally  be  cultivated  under  saprophytic 
conditions,  and  the  vast  majority  of  other  forms  live  as 
true  saprophytes  on  dead  organic  matter  wherever  it 
may  occur  in  nature,,  and  particularly  in  that  diffuse  layer 
of  decomposing  plant  and  animal  material  which  we  call 
the  humus,  or  surface  layer  of  the  soil.  Wherever  there 
is  life,  waste  matter  is  constantly  being  produced,  and 
this  finds  its  way  to  the  earth  or  to  some  body  of  water. 
The  excretions  of  animals,  the  dead  tissues  and  broken- 
down  cells  of  both  animals  and  plants,  as  well  as  the 
wastes  of  domestic  and  industrial  life,  all  eventually  find 
their  way  to  the  soil.  In  a  majority  of  cases  these  sub- 
stances are  not  of  such  chemical  composition  that  they 
can  be  utilized  at  once  by  green  plants  as  food,  but  it  is 
first  necessary  that  they  go  through  a  fermentation  or  trans- 
formation in  which  their  chemical  composition  becomes 
greatly  changed;  and  it  is  as  the  agents  of  this  trans- 
formation that  bacteria  assume  their  greatest  importance 
in  the  world  of  life. 


THE  BACTERIA   IN  NATURAL    WATERS.  5 

We  may  take  a  comparatively  simple  excretory  prod- 
uct, urea,  as  an  example.  Through  the  activity  of  an 
enzyme  produced  by  certain  bacteria  this  compound 
unites  with  two  molecules  of  water  and  is  converted  into 
ammonium  carbonate, 

/NH2 
C0 


While  green  plants  can  derive  their  necessary  nitrogen 
in  part,  at  least,  from  ammonium  compounds  it  is  a  well- 
established  fact  that  this  element  may  be  obtained  much 
more  readily  from  nitrates,  and  it  is,  therefore,  essential 
as  a  further  step  that  some  means  be  employed  to  oxidize 
the  nitrogen.  This  process  of  oxidation  is  known  as 
nitrification,  and  takes  place  in  a  succession  of  steps, 
the  organic  nitrogen  being  first  converted  to  the  form  of 
ammonium  salts,  and  these  in  turn  to  nitrites  and  nitrates, 
the  oxygen  used  coming  from  the  air.  Several  groups 
of  organisms  are  instrumental  in  bringing  about  this  con- 
version. It  is  generally  assumed  that  one  group  attacks 
the  ammonium  compounds  and  changes  them  to  nitrites, 
while  another  group  completes  the  oxidation  to  nitrates. 
In  the  latter  form  nitrogen  is  readily  taken  up  by  green 
plants  to  be  built  up  into  the  more  complex  albuminoid 
substances  (organic  nitrogen)  through  the  constructive 
power  of  chlorophyll. 

This  never-ending  cycle  is  illustrated  in  the  accom- 
panying figure,  devised  by  Sedgwick  (Sedgwick,  1889) 
to  illustrate  the  transformations  of  organic  nitrogen  in 


6  ELEMENTS   OF   WATER  BACTERIOLOGY. 

nature,  the  increasing  size  and  closeness  of  the  spiral  on 
the  left-hand  side  indicating  the  progressive  complexity 
of  organic  matter  as  built  up  by  the  chlorophyll  bodies 
of  green  plants  in  the  sunlight,  and  the  other  half  of  the 
figure  the  reverse  process  carried  out  largely  by  the  bac- 
teria. In  nature  there  are  many  short  circuits,  as,  for 
instance,  when  dead  organic  matter  is  used  as  food  for 
animals  and  built  up  into  the  living  state  again  without 
ever  being  nitrified  and  acted  upon  by  green  plants;  but 
the  complete  cycle  of  organic  nitrogen  is  as  indicated  on 
the  diagram. 

We  have  dwelt  thus  at  length  upon  the  general  relation 
between  bacteria  and  organic  decomposition  because  in 
this  relation  will  be  found  the  master  key  to  the  distri- 
bution of  bacteria  in  water  as  well  as  in  other  natural 
habitats.  It  is  true  that  certain  peculiar  forms  may  at 
times  multiply  in  fairly  pure  waters;  but  in  general  large 
numbers  of  bacteria  are  found  only  in  connection  with 
the  organic  matter  upon  which  they  feed.  Such  organic 
matter  is  particularly  abundant  in  the  surface  layer  of  the 
soil.  Here  the  processes  of  nitrification  proceed  most 
rapidly.  Here  the  bacteria  are  most  abundant;  and  in 
other  media  their  numbers  vary  according  to  the  extent 
of  contact  with  the  living  earth.  Natural  waters  particu- 
larly group  themselves  from  a  bacteriological  standpoint 
in  three  well-marked  classes,  according  to  their  relation 
to  the  rich  layers  of  bacterial  growth  upon  the  surface  of 
the  globe.  There  are  first  the  atmospheric  waters  which 
have  never  been  subject  to  contact  with  the  earth;  second, 


THE  BACTERIA   IN  NATURAL    WATERS. 


7 


the  surface-waters  immediately  exposed  to  such  contami- 
nation in  streams  and  ponds;  third,  the  ground- waters 
from  which  previous  pollution  has  been  more  or  less 
removed  by  filtration  through  the  deeper  layers  of  the 
soil. 

Even  rain  and  snow,  the  sources  of  our  potable  waters, 
are  by  no  means  free  from  germs,  but  contain  them  in 
numbers  varying  according  to  the  amount  of  dust  present 


NITROGEN  AS 
ORGANIC  NITROGEN 

(ALBUMINOID  AMMONIA) 


in  the  air  at  the  time  of  the  precipitation.  After  a  long- 
continued  storm  the  atmosphere  is  washed  nearly  free 
of  bacteria,  so  that  a  considerable  series  of  sterile  plates  may 
often  be  obtained  by  plating  i-c.c.  samples.  These  re- 
sults are  in  harmony  with  the  observations  of  Tissandier 
(reported  by  Duclaux,  1897),  wno  found  that  the  dust  in 
the  air  amounted  to  23  mg.  per  cubic  meter  in  Paris 


8  ELEMENTS   OF   WATER  BACTERIOLOGY. 

and  4  mg.  in  the  open  country.  After  a  rainfall  these 
figures  were  reduced  to  6  mg.  and  .25  mg.,  respectively. 

With  regard  to  what  may  be  considered  normal  values 
for  rain  we  have  no  very  satisfactory  figures.  Those 
obtained  by  Miquel  (Miquel,  1886)  during  the  period 
1883-1886,  showing  that  rain  contains  on  the  average  4.3 
bacteria  per  c.c.  in  the  country  (Montsouris)  and  19  per 
c.c.  in  Paris,  are  probably  lower  than  would  be  yielded  by 
the  present  methods  of  examination.  Snow  shows  rather 
higher  numbers  than  rain.  Janowski  (Janowski,  1888) 
found  in  freshly  fallen  snow  from  34  to  463  bacteria  per 
c.c.  of  snow-water,  and  his  results  indicate  that  the  num- 
ber is  independent  of  the  temperature  at  the  time  of 
snowfall. 

As  soon  as  the  rain- drop  touches  the  surface  of  the 
earth  its  real  bacterial  contamination  begins.  Rivulets 
from  ploughed  land  or  roadways  may  often  contain  several 
hundred  thousand  bacteria  to  the  cubic  centimeter;  and 
furthermore  the  amounts  of  organic  and  mineral  mat- 
ters which  serve  as  food  materials,  and  thus  become  a 
factor  in  later  multiplication  of  organisms,  are  greatly 
increased. 

In  the  larger  streams  several  conditions  combine  to 
make  the  bacterial  numbers  lower.  Ground-water  con- 
taining little  microbic  life  enters  as  a  diluting  factor  from 
below.  The  larger  particles  of  organic  matter  are  re- 
moved from  the  flowing  water  by  sedimentation;  many 
earth  bacteria,  for  which  water  is  an  unfavorable  medium, 
gradually  perish ;  and  in  general  a  new  condition  of  equi- 


THE  BACTERIA   IN  NATURAL    WATERS.  g 

librium  tends  to  be  established.  A  good  river- water  under 
favorable  conditions  should  thus  contain  only  a  few  hun- 
dred bacteria.  Heavy  rains  which  introduce  wash  from 
the  surrounding  watershed  may,  however,  at  any  time 
upset  this  condition  of  equilibrium  and  surface-waters 
are  apt  to  show  sudden  fluctuations  in  their  bacterial  con- 
tent. Particularly  in  the  spring  and  fall  high  numbers 
manifest  themselves,  and  seasonal  variations  arise,  such 
as  are  well  shown  in  the  appended  table. 


NUMBER  OF   BACTERIA   PER    CUBIC    CENTIMETER   IN   CERTAIN 
SURFACE-WATERS. 


Water. 

Yr. 

Observer. 

Jan. 

Feb. 

March. 

April. 

May. 

June 

Boston   tap  j 
water  J 

1892- 
1895 

j-  Whipple* 

135 

211 

102 

52 

53 

86 

Merrimac  River  . 

1901 

Clark  t 

5,6oc 

2,500 

8,900 

1,400 

1,400 

3000 

Thames  River.  .  . 

1888 

FranklandJ 

92,000 

40,000 

66,000 

13,000 

1,900 

35oo 

River  Ourcq.  .  -j 

1887- 
1890 

j-  Miquel  § 

I43.37C 

63,720 

47,78o 

22,660 

29,340 

7340 

Water. 

Yr. 

Observer. 

July. 

A.ug. 

Sept. 

Oct. 

Nov. 

Dec. 

Boston   tapj 
water  I 

1892- 
J895 

j-  Whipple* 

73 

81 

86 

55 

56 

52 

Merrimac  River. 

1901 

Clarkf 

1760 

1580 

1760 

1,900 

1,300 

5,ioo 

Thames  River.  .  . 

1888 

FranklandJ 

1070 

3000 

1740 

1,130 

11,700 

10,600 

River  Ourcq  .  .  -j 

1887- 
1890 

j-  Miquel  § 

7730 

8520 

8070 

12,560 

i35,7oo 

153,200 

*  Whipple,  1896. 
t  Frankland,  1894. 


t  Massachusetts  State  Board  of  Health,  1902. 
§  Miquel,  1891. 


•  Two  factors  influence  this  seasonal  distribution  of  bac- 
teria. First,  during  the  summer  months  the  water  flow-  i 
ing  in  open  rivers  is  largely  derived  from  springs  and 
subterranean  sources,  while  during  the  autumn  and 
spring  months,  there  is  a  much  greater  proportion  of 
"  run-off "  water  contaminated  by  contact  with  the  sur- 


IO 


ELEMENTS  OF   WATER  BACTERIOLOGY. 


face  of  the  earth.  In  the  second  place,  during  these 
rainy  seasons  the  amount  of  dissolved  organic  matter  is 
far  greater  than  in  summer,  thus  making  the  food-supply 
of  the  bacteria  more  abundant. 

In  standing  waters  all  the  tendencies  which  make  for 
the  reduction  of  bacteria  are  intensified,  and  ponds  and 
lakes  often  give  numbers  under  a  hundred.  The  student 
will  find  numerous  analyses  of  natural  waters  in  Frank- 
land's  classic  work  (Frankland,  1894).  He  notes,  for 
example,  that  the  Lake  of  Lucerne  contained  8  to  5 1  bac- 
teria per  c.c.,  Loch  Katrine  74,  and  the  Loch  of  Lintral- 
then  an  average  of  170.  The  water  of  Lake  Champlain 
examined  by  one  of  us  (S.  C.  P.)  in  1896  contained  on 
an  average  82  bacteria  per  c.c.  at  a  point  more  than  two 
miles  out  from  the  city  of  Burlington.  Certain  surface 
water-supplies  near  Boston  studied  by  Nibecker  and  one 
of  us  (Winslow  and  Nibecker,  1903)  gave  the  following 
results : 


City. 

Number  of 
Samples. 

Average 
Number 
of  Bacteria 
per  cc. 

Wakefield 

7" 

CQ 

Lynn  

6 

16 

Plymouth 

6 

•?(? 

Cambridge 

c 

04. 

Salem 

2"?2 

Medford. 

CT24 

Taunton.  .                  

4 

12 

Peabody  

•3 

141 

Russell  found  similar  small  numbers  in  sea-water  at 
Naples  (Russell,  1891)  and  Wood's  Hole  (Russell,  1892), 


THE  BACTERIA  IN  NATURAL   WATERS.  II 

and  in  salt  as  in  fresh  water  the  amount  of  bacterial  life 
decreases  in  general  as  one  passes  downward  from  the 
surface  and  outward  from  the  shore. 

The  principal  factors  in  the  destruction  of  the  bacteria 
in  water  during  storage  appear  to  be  sedimentation,  the 
activity  of  other  micro-organisms,  light,  temperature,  and 
food-supply,  and  perhaps  more  obscure  conditions  such 
as  osmotic  pressure. 

The  subsidence  of  bacteria  either  by  virtue  of  their  own 
specific  gravity  or  as  the  result  of  becoming  attached  to 
particles  of  suspended  matter  is  unquestionably  partly, 
if  not  largely,  responsible  for  changes  in  the  number  of 
bacteria  in  the  upper  layers  of  water  at  rest  or  in  very 
sluggish  streams.  The  results  of  numerous  inves- 
tigations by  different  workers  seem  to  indicate  that 
sedimentation  takes  place  slowly,  and  that  the  differ- 
ence in  numbers  between  the  top  layer  and  the  bottom 
layer  of  water  in  tall  jars  in  laboratory  experiments  of 
only  a  few  days'  duration  is  very  slight  or  quite  within 
the  limits  of  experimental  error  (Tiemann  and  Gartner, 
1889).  Different  species  may,  of  course,  be  differently 
affected,  (Scheurlen,  1891).  It  must  be  remembered, 
that  in  natural  streams  bacteria  are  to  a  great  extent 
attached  to  larger  solid  particles  upon  which  the  action 
of  gravity  is  much  more  important.  Jordan  (Jordan, 
1900)  is  firmly  of  the  opinion  that  in  the  lower  part 
of  the  Illinois  River,  where  there  is  a  fall  of  but  30 
feet  in  225  miles,  the  influences  summed  up  by  the  term 
sedimentation  are  sufficiently  powerful  to  obviate  the 


12  ELEMENTS   OF  WATER  BACTERIOLOGY. 

necessity  for  summoning  another  cause  "to  explain  the 
diminution  in  numbers  of  bacteria,"  and  he  further  adds: 
11  It  is  noteworthy  that  all  the  instances  recorded  in  the 
literature  where  a  marked  bacterial  purification  has  been 
observed  are  precisely  those  where  the  conditions  have 
been  most  favorable  for  sedimentation." 

Little  is  known  as  to  the  share  of  other  organisms  in 
hastening  the  decrease  of  bacteria  in  stored  water.  Doubt- 
less predatory  Protozoa  play  some  part  here,  and  cer- 
tain bacteriologists  have  believed  that  the  toxic  waste 
products  of  some  species  of  bacteria  materially  check 
the  development  of  other  forms.  Horrocks  (Horrocks, 
1901),  Garre  (Garre,  1887),  Zagari  (Zagari,  1887), 
and  Freudenreich  (Freudenreich,  1888)  have  shown 
that  an  "  antagonism"  exists  when  bacteria  are  grown 
in  artificial  culture  media  such  that  the  substratum 
which  has  supported  the  growth  of  one  form  may  be 
rendered  antiseptic  to  another.  It  is  difficult,  however, 
to  believe  that  any  poisons  are  produced  of  such  enor- 
mous power  as  to  cause  this  effect  in  a  stream  or  a 
lake,  and  there  is  no  evidence  in  support  of  such  a 
view. 

Temperature  has  a  direct  relation  to  bacterial  life,  and 
the  number  of  parasitic  bacteria  at  least  may  be  quickly 
lessened  by  the  action  of  cold.  Sedgwick  and  one  of  us 
(Sedgwick  and  Winslow,  1902)  have  shown  that  of 
typhoid  bacilli  in  ice  or  cool  water  over  40  per  cent  will 
perish  in  three  hours  and  98  per  cent  and  upwards  in  two 
weeks. 


THE 


IN  NATURAL    WATERS.  13 

Many  investigations  conducted  since  the  pioneer  re- 
searches of  Downes  and  Blunt  (Downes  and  Blunt,  1877) 
have  confirmed  the  results  reported  by  them,  viz.,  direct 
sunlight  is  fatal  to  most  bacteria  in  the  vegetative  state 
and  even  to  spores  if  the  exposure  be  sufficiently  long, 
while  diffused  light  is  harmful  in  a  lesser  degree.  Opin- 
ions vary  as  to  the  degree  to  which  light  is  active  in 
destroying  the  bacteria  in  natural  water.  Buchner 
(Buchner,  1893)  found  by  experiment  that  the  bacteri- 
cidal power  of  light  extends  to  a  depth  of  about  three 
meters  before  it  becomes  imperceptible.  On  the  other 
hand,  Procaccini  (Procaccini,  1893)  found  that  when  sun- 
light was  passed  vertically  through  60  cm.  of  drain-water 
the  lower  layers  contained  nearly  as  many  bacteria  after 
three  hours'  treatment  as  before  the  exposure.  The 
middle  and  upper  portions  showed  a  great  falling  off  in 
numbers,  however. 

But  few  studies  have  been  made  of  the  effect  of  light  on 
bacteria  in  flowing  water.  Jordan  (Jordan,  1900)  has 
investigated  several  Illinois  streams  and  arrived  at  the 
conclusion  that  in  moderately  turbid  water,  at  least,  the 
sun's  rays  are  virtually  without  action.  On  the  other 
hand,  Rapp  has  observed  a  considerable  reduction  of 
the  bacteria  in  the  Isar  at  Pullach  after  the  period  of 
diurnal  insolation,  as  shown  by  the  table  on  the  following 
page. 

Although  it  is  hard  to  estimate  the  exact  importance 
of  each  factor,  the  general  phenomena  of  the  self-purifi- 
cation of  streams  are  easy  to  comprehend.  A  small 


ELEMENTS   OF   WATER  BACTERIOLOGY. 


EXAMINATIONS  or  THE  ISAR  AT  PULLACH  (RAPP,  1903). 

(A)    CARRIED  OUT  SEPTEMBER  26,    1898,   NO  RAIN  HAVING  FALLEN    FOR 
THREE   WEEKS. 


Temperature 

Time  of  the 

Bacteria 

Experiment. 

per  cc. 

of  the  Water. 

of  the  Air. 

13.0°  C. 

8.8°  C. 

7.30  P.M. 

146 

12.1°  C. 

7.0°  C. 

9.30  P.M. 

270 

10.5°  C. 

6.2°  C. 

5-00  A.M. 

37° 

10.2°  C. 

8.2°  C. 

8.00  A.M. 

320 

(B)     CARRIED  OUT    NOVEMBER   28,    1898,   NO  RAIN  HAVING  FALLEN  FOR 
SOME   TIME. 


5-5°  C. 

3-o°  C. 

6.00  P.M. 

266 

5-5°  C. 

2.5°  c. 

8.00  P.M. 

402 

5-5°  C. 

2.0°  C. 

10.00  P.M. 

482 

5-o°  C. 

2.0°  C. 

3.OO  A.M. 

532 

4.5°  C. 

2.5°  C. 

7.30  A.M. 

40O 

brook  immediately  after  the  entrance  of  polluting  mate- 
rial from  the  surface  of  the  ground  contains  a  large  num- 
ber of  bacteria  from  a  diversity  of  sources.  Gradually 
those  organisms  adapted  to  life  in  the  earth  or  in  the 
bodies  of  plants  and  animals  die  out,  and  the  forms 
for  which  water  furnishes  ideal  conditions  survive 
and  multiply.  It  is  no  single  agent  which  brings  this 
about,  but  that  complex  of  little-understood  conditions 
which  we  call  the  environment.  If  any  one  thing  is  of 
prime  importance  it  is  probably  the  food-supply,  for  only 
certain  bacteria  are  able  to  multiply  in  the  presence  of  the 
small  amount  of  organic  matter  present  in  ordinary  po- 
table waters.  vAs  Jordan  (Jordan,  1900)  has  said:  "In 
the  causes  connected  with  the  insufficiency  or  unsuitability 


THE   BACTERIA   IN  NATURAL    WATERS.  15 

of  the  food- supply  is  to  be  found,  I  believe,  the  main  rea- 
son for  the  bacterial  self-purification  of  streams." 

In  general  we  have  seen  then  that  surface-waters  tend 
continually  to  decrease  in  bacterial  content  after  their 
first  period  of  contact  with  the  humus  layer  of  the  soil. 
In  that  other  portion  of  the  meteoric  water  which  pene- 
trates below  the  surface  of  the  earth  to  join  the  reservoir 
of  ground-water,  later  to  reappear  as  the  flow  of  springs 
and  wells,  this   diminution  is  still  more  marked   since 
the  filtering  action  of  the  earth  removes  not  only  most  ctf 
of  the  bacteria  but  much*of  their  food  material  as  well,  ^j 
Indeed  many  observers  formerly  believed  that  all  ground-  ;; 
waters  were  nearly  free  from  bacteria,  because  often  no  *-* 
colonies  appeared  on  plates  counted  after  the  ordinary  : : 

I  ~j 

short  periods  of  time.  If,  however,  a  longer  period  of  ^ 
incubation  be  adopted  considerable  numbers  may  be  ^ 
obtained. 

For  convenience  we  may  divide  ground- waters  into  two  W 
groups,  namely:  first,  springs  and  shallow  open  wells,  andS 
second,  "tubular"  (driven)  or  deep  wells.  This  division  i 
a  convenient  one  because  ordinary  springs  and  wells  form 
a  group  by  themselves  in  respect  to  the  possibility  of 
aerial  and  surface  contamination,  their  water  often  being 
fairly  rich  in  bacterial  life.  Egger  (Wolffhugel,  1886)  ex- 
amined 60  wells  in  Mainz  and  found  that  17  of  them 
contained  over  200  bacteria  to  the  cubic  centimeter. 
Maschek  (Maschek,  1887)  found  36  wells  out  of  48  exam- 
ined in  Leitmeritz  which  had  a  bacterial  content  of  over 
500  per  c.c.  Fischer  (Horrocks,  1901)  reported  120  wells 


i6 


ELEMENTS  OF  WATER  BACTERIOLOGY. 


in  Kiel  which  gave  over  500  bacteria  per  c.c.  and  only  51 
with  less  than  that  number. 

Several  unpolluted  springs  and  open  wells  were  ex- 
amined by  Sedgwick  and  one  of  us  (Sedgwick  and  Pres- 
cott,  1895)  with  the  following  result: 

Spring  No.  i.  252,  255,  258  bacteria  per  c.c. 

"      2.    163,     149,     134 

'    3-     92,  98,  105 

"        "    4-    95>  101,  106 

"        "    5.  193,  213,  218 

"        "6.  216,  208,  201 

OPEN   WELLS. 

No.  i.     509,  525  bacteria  per  c.c. 

"  2.     248,  190        " 

"  3-    602,  560  " 

"  4-     335>  332  " 

"  5.  2084,  2063,  2287  bacteria  per  c.c. 

"  6.  8905,  8905,  8640 

"  7.     702,     910,  871 

"  8.    720,     712,  763 

It  should  be  noted  that  the  above  results  were  obtained 
by  incubating  the  plates  for  considerable  periods  of  time. 
In  the  ordinary  standard  48-hour  period  but  very  few 
bacteria  develop  from  normal  ground-waters.  Thus  in 
an  examination  of  spring-waters  made  by  the  Massa- 
chusetts State  Board  of  Health  in  1900  (Massachusetts 
State  Board  of  Health,  1901),  of  37  springs  which  were 
practically  unpolluted  and  had  less  than  10  parts  per 
100,000  excess  of  chlorine  over  the  normal,  54  samples 
were  examined  and  gave  an  average  of  41  bacteria  per 
c.c.  Only  6  samples  showed  figures  over  50.  On  the 
other  hand,  the  analysis  of  the  bottled  samples  of  the 
same  waters  as  sold  after  exposure  to  contamination  in 


THE  BACTERIA  IN*  NATURAL    WATERS.  17 

bottling  and  the  multiplication  of  the  water  bacteria 
gave  numbers  rising  to  243,000,  only  14  out  of  50  samples 
being  under  1000. 

It  now  remains  to  consider  the  other  great  division  of 
ground- waters,  namely,  deep,  " driven,"  or  "tubular" 
wells,  which,  if  carefully  constructed,  should  ordinarily  be 
perfectly  free  from  all  surface-water  contamination.  The 
numbers  of  bacteria  in  such  sources  has  been  reported 
by  but  few  investigators.  A  series  of  wells  in  and  near 
Boston  was  found  to  give  the  following  figures  (Sedgwick 
and  Prescott,  1895). 

Well.                                                    Depth,  Feet.  Bacteria  per  c.c. 

No.     1 193  269,254 

"         2 100  30 

"       3 454  206,214 

"       4 254  I50.I3S 

"      5 228 

"      6 198  192,  193 

Second  sample 262,  258 

"       7 213  139.140 

8 213  101,106 

Second  sample 408,  416 

"      9 377  48,    54 

Second  sample 158,  149 

10 227  1240,1376 

ii 130  440,480 

"       12 200  525 

"     i3 180  60,    57 

M 750  38 

Again  it  should  be  noted  that  the  period  of  incubation 
for  these  samples  was  at  least  five  days.  Fifteen  driven 
wells  in  the  neighborhood  of  Boston  examined  in  1903 
showed  at  the  end  of  48  hours  an  average  of  only  18 
colonies  per  c.c. 


iS  ELEMENTS   OF  WATER  BACTERIOLOGY. 

It  is  plain  that  water  absolutely  free  from  bacteria  is 
not  ordinarily  obtained  from  any  source  and  that  even 
deep  wells  contain  quite  appreciable  numbers.  The 
peculiar  character  of  the  organisms  present  in  the  latter 
case  is  manifested  in  many  cases  by  the  slow  develop- 
ment at  room  temperature  (no  growth  until*  the  third 
day  in  some  cases),  the  entire  absence  of  liquefying 
colonies,  and  the  abundance  of  chromogenic  species. 


CHAPTER  II. 

THE  QUANTITATIVE  BACTERIOLOGICAL  EXAMINATION 
OF  WATER. 

THAT  the  customary  methods  for  determining  the  num- 
ber of  bacteria  do  not  reveal  the  total  bacterial  content, 
but  only  a  very  small  fraction  of  it,  becomes  apparent 
when  we  consider  the  large  number  of  organisms,  nitrify- 
ing bacteria,  cellulose- fermenting  bacteria,  strict  anae- 
robes, etc.,  which  refuse  to  grow,  or  grow  only  very  slowly 
in  ordinary  culture  media,  and  which,  therefore,  escape 
our  notice.  On  the  one  hand  certain  obligate  parasites 
cannot  thrive  in  the  absence  of  the  rich  fluids  of  the  ani- 
mal body;  on  the  other  hand  the  prototrophic  bacteria 
adapted  to  the  task  of  wrenching  energy  from  nitrates 
and  ammonium  compounds  are  unable  to  develop 
in  the  presence  of  so  much  organic  matter.  This 
is  made  clear  by  the  use  of  special  media  like  the 
NahrstofT  Heyden  agar  (Hesse  and  Niedner,  1898), 
which  are  particularly  adapted  to  the  needs  of  these 
latter  organisms;  or  by  direct  microscopic  examin- 
ation. Thus  Gage  and  Phelps  (Gage  and  Phelps, 

Note.    In  this  chapter  the  authors  have  closely  followed  the  recom- 
mendations of  the  A.  P.  H.  A.,  as  given  in  the  Appendix. 

19 


20  ELEMENTS  OF   WATER  BACTERIOLOGY. 

1902)  showed  that  the  numbers  obtained  by  the  ordi- 
nary procedure  were  only  from  5  to  50  per  cent  of 
those  obtained  by  the  use  of  Heyden's  Nahrstoff  agar. 
For  practical  sanitary  purposes,  however,  our  methods  are 
fairly  satisfactory.  Within  limits,  it  is  of  no  great  impor- 
tance that  one  method  allows  the  growth  of  more  bacteria 
than  another.  When  we  are  using  the  quantitative  analy- 
sis as  a  measure  of  sewage  pollution  only  two  things  are 
essential.  First,  media  should  be  of  standard  composi- 
tion, so  that  results  obtained  at  different  times  and  by 
different  observers  may  be  comparable.  In  this  respect 
the  work  of  G.  W.  Fuller,  G.  C.  Whipple,  and  other 
members  of  the  Committee  on  Standard  Methods  of  the 
American  Public  Health  Association  has  placed  the  art  of 
quantitative  water  analysis  in  a  state,  very  satisfactory,  by 
contrast  with  the  chaos  which  prevails  in  England  and 
Germany.  Secondly,  it  is  desirable  that  the  section  of 
the  total  bacterial  flora  which  we  obtain  should  be  thor- 
oughly representative  of  that  portion  of  it  in  which  we 
are  most  interested — the  group  of  the  quickly  growing, 
rich-food-loving  sewage  forms.  In  this  respect  our  meat- 
gelatin-peptone  appears  to  be  unrivalled.  The  follow- 
ing table  from  Gage  and  Phelps's  valuable  paper  shows 
clearly  that  the  standard  media  bring  out  the  difference 
between  pure  and  polluted  waters  much  more  clearly  than 
does  the  Nahrstoff  medium.  To  emphasize  this  difference 
with  constancy  is  all  that  we  require  of  a  method  for  prac- 
tical work. 


QUANTITATIVE  BACTERIOLOGICAL  EXAMINATION.    21 


TABLE  SHOWING  PERCENTAGES  OF  BACTERIA  DEVELOPING  ON  REGU- 
LAR AGAR  AND  ON  NAHRSTOFF  AGAR  FOR  DIFFERENT  CLASSES  OF 
WATERS.  (GAGE  AND  PHELPS,  1902.) 

REGULAR  AGAR. 


Class  of  Water. 

Days'  Count. 

2 

3 

4 

5 

6 

7 

8 

Ground-water 

o 
6 
6 
14 
34 

5 
7 
7 
i7 
44 

6 

7 

18 

46 

6 

8 

19 
46 

6 

I 

iQ 
46 

6 
7 
9 
19 
46 

6 
7 
9 
J9 
46 

Filtered  water                                  .    . 

Merrirnac  River.             

Filtered  sewage  

NAHRSTOFF  AGAR. 


G  round-water 

6 

4.3 

78 

88 

Q5 

IOO 

IOO 

Filtered  water 

•37 

DO 

80 

02 

08 

IOO 

IOO 

\lerrimac  River 

20 

78 

Q7 

07 

O7 

OO 

IOO 

Filtered  sewage 

26 

*5 

Q2 

QC 

O7 

OO 

IOO 

Sewage  .                         

•2Q 

7e 

QC 

IOO 

IOO 

IOO 

IOO 

The  procedure  for  the  quantitative  determination  of 
bacteria  in  water  consists,  in  brief,  in  mixing  a  definite 
amount  of  a  suitably  collected  specimen  of  the  water  with 
a  sterile  solidifiable  culture  medium  and  allowing  it  to 
develop  for  a  sufficiently  long  time  to  permit  reproduction 
of  the  bacteria  and  the  formation  of  visible  colonies  which 
may  be  counted.  The  process  is  divided  naturally  into 
four  stages — sampling,  plating,  incubating,  and  counting 
— which  will  now  be  discussed. 

Sampling. — All  samples  of  water  for  bacteriological 
examination  should  be  collected  in  clean,  sterile  bottles 
with  wide  mouths  and  glass  stoppers,  preferably  of  the  flat 


22  ELEMENTS   OF  WATER  BACTERIOLOGY. 

mushroom  type.  It  is  desirable  that  these  bottles  should 
have  a  capacity  of  at  least  100  c.c. 

They  should  be  cleaned  thoroughly  before  using,  by 
treatment  with  sulphuric  acid  and  potassium  bichromate 
or  with  alkaline  permanganate  of  potash  followed  by 
sulphuric  acid,  dried  by  draining,  and  sterilized  by  dry 
heat  at  160°  C.  for  at  least  one  hour,  or  by  steam  at  115°- 
120°  for  fifteen  minutes.  If  not  to  be  used  immediately 
the  neck  and  stopper  should  be  protected  against  dust 
or  other  contamination  by  wrapping  with  lead-foil.  For 
transportation  the  bottle  should  be  enclosed  in  a  suitable 
case  or  box. 

The  greatest  care  must  be  taken  that  the  fingers  do  not 
touch  the  inside  of  the  neck  of  the  bottle  or  the  cone  of  the 
stopper,  as  the  water  thereby  would  become  seriously 
contaminated  and  rendered  unfit  for  examination.  It  is 
well  known  that  bacteria  are  found  abundantly  upon 
the  skin,  and  Winslow  (Winslow,  1903)  has  shown  that 
even  B.  coli  is  present  upon  the  hands  in  a  considerable 
number  of  cases. 

In  order  to  obtain  a  fair  sample,  great  precautions  must 
be  taken,  and  these  will  vary  with  the  different  classes  of 
waters  to  be  examined  and  with  local  conditions.  If  a 
sample  is  to  be  taken  from  a  tap,  the  water  should  be 
allowed  to  flow  at  least  five  minutes  (if  from  a  tap  in  regu- 
lar use)  or  for  a  longer  period  in  case  the  water  has  been 
standing  in  the  house  service  system,  since  in  the  small 
pipes,  changes  in  bacterial  content  are  liable  to  occur, 


QUANTITATIVE  BACTERIOLOGICAL  EXAMINATION.    23 

certain  species  dying,  and  the  sample,  therefore,  not  being 
a  fair  average  of  the  water  in  the  mains. 

If  a  sample  is  to  be  taken  from  a  pump  similar  pre- 
cautions are  necessary.  The  pump  should  be  in  con- 
tinuous operation  for  several  minutes  at  least,  and  prefer- 
ably for  half  an  hour  before  the  sample  is  taken,  in  order 
to  avoid  excessively  high  numbers  due  to  the  growth  of 
bacteria  within  the  well  and  pump,  the  bacterial  condition 
of  the  water  as  it  passes  through  the  ground  being  what 
we  wish  to  determine.  Thus  Heraeus  (Heraeus,  1886)  in 
a  well-water  which  had  been  but  little  used  during  the 
preceding  thirty-six  hours  found  5000  organisms  per  c.c.; 
when  the  well  was  emptied  by  continuous  pumping  a 
second  sample,  after  an  interval  of  half  an  hour,  gave  only 
35.  Maschek  (Tiemann  and  Gartner,  1889)  obtained 
similar  results  shown  in  the  following  table: 

EFFECT   OF  PUMPING  ON  THE   BACTERIAL   CONTENT   OF   WELL-WATER. 

Well-water  after  continuous  pumping  for  fifteen  minutes 458 

"            "                "         "  many  hours 140 

later 68 

after  continuous  pumping  for  fifteen  minutes 578 

"            "                "          "     many  hours 179 

later 73 

After  a  proper  interval  of  pumping  the  sample  of  a  well- 
water  may  be  collected  from  the  pet- cock  of  the  pump  or 
from  a  near-by  tap.  With  a  hand-pump,  as  in  sampling 
domestic  shallow  wells,  the  water  is,  of  course,  pumped 
directly  into  the  sample  bottle.  The  difficulties  in 
securing  an  average  sample  from  this  latter  source  are 


24  ELEMENTS   OF   WATER  BACTERIOLOGY. 

often  great,  since  if  the  flooring  about  the  pump  is  not 
tight,  as  is  usually  the  case,  continued  pumping  may  wash 
in  an  unusual  amount  of  surface  pollution. 

In  sampling  surface-waters,  the  greatest  precautions 
must  be  observed  to  prevent  contamination  from  the 
fingers.  In  still  waters  the  fairest  sample  is  one  taken 
from  several  inches  down,  as  the  surface  itself  is  likely  to 
have  numerous  dust  particles  floating  upon  it.  The 
method  most  frequently  recommended  is  to  plunge  the 
bottle  beneath  the  surface  to  a  depth  of  a  foot  or  so,  then 
remove  the  stopper  and  allow  the  bottle  to  fill. 

Another  method  which  is  comparatively  free  from 
objection  and  which  has  been  employed  by  the  writers  is 
to  remove  the  stopper  first  and  then,  holding  the  bottle  by 
the  base,  plunge  it  mouth  downward  into  the  water,  turn- 
ing it  at  the  desired  depth  so  as  to  replace  the  enclosed 
air  by  the  water.  Whenever  any  current  exists  the 
mouth  of  the  bottle  should  be  directed  against  it  in 
order  to  carry  away  any  bacteria  from  the  fingers.  If 
there  is  no  current,  a  similar  effect  can  be  produced  by 
turning  the  bottle  under  water  and  giving  it  a  quick  for- 
ward motion.  In  rapidly  flowing  streams  it  is  only 
necessary  to  hold  the  bottle  at  the  surface  with  the  mouth 
pointed  up-stream. 

For  taking  samples  of  water  at  greater  depths,  a  num- 
ber of  devices  have  been  employed,  all  of  which  are  fairly 
satisfactory.  The  essentials  are,  first,  a  weight  to  carry 
the  bottle  down  to  the  desired  depth,  and,  second,  a  device 


QUANTITATIVE  BACTERIOLOGICAL  EXAMINATION.   2$ 

for  removing  the  stopper  when  that  depth  is  reached.  The 
student  will  find  one  good  form  of  apparatus  described 
in  Abbott's " Principles  of  Bacteriology"  (Abbott,  1899); 
an  admirable  one  was  devised  by  Hill  and  Ellms  (Hill 
and  Ellms,  1898).  Miquel  and  Cambier  (Miquel  and 
Cambier,  1902)  and  other  authors  recommend  the  use  of 
a  sealed  glass  bulb  with  a  capillary  tube  which  can  be 
broken  off  at  any  desired  moment. 

As  soon  as  a  sample  of  water  is  collected  its  conditions 
of  equilibrium  are  upset  and  a  change  in  the  bacterial 
content  begins.  Even  in  the  purest  spring-waters  which 
contain  but  few  bacteria  when  collected,  and  in  which  the 
amount  of  organic  matter  is  infinitesimal,  enormous  num- 
bers will  be  found  after  storage  under  laboratory  con- 
ditions for  a  few  days  or  even  a  few  hours.  In  some  cases 
the  rise  in  numbers  is  gradual,  in  others  very  rapid.  The 
Frariklands  (Frankland,  1894)  record  the  case  of  a  deep- 
well  water  in  which  the  bacteria  increased  from  7  to 
495,000  in  three  days.  Miquel  (Miquel,  1891),  from  his 
researches,  arrived  at  the  conclusion  that  in  surface-waters 
the  rise  is  less  rapid  than  in  waters  from  deep  wells  or 
springs,  and  that  in  the  latter  case  the  decrease,  after 
reaching  a  maximum,  is  likewise  rapid  and  steady.  Just 
how  far  protection  from  light,  increase  in  temperature, 
and  a  destruction  of  higher  micro-organisms  is  responsible 
for  the  increase,  and  to  what  extent  an  exhaustion  of  food- 
supply  or  the  formation  of  toxic  waste  products  causes 
the  succeeding  decrease,  we  are  not  aware;  but  the  facts 
are  well  established. 


26 


ELEMENTS  OF   WATER  BACTERIOLOGY. 


Whipple  has  exhaustively  studied  the  details  of  this 
multiplication  of  bacteria  in  stored  waters  and  has  shown 
in  the  table  given  below  that  there  is  first  a  slight  reduc- 
tion in  the  number  present,  lasting  perhaps  for  six  hours, 
followed  by  the  great  increase  noted  by  earlier  observers. 
It  is  probable  that  there  is  a  constant  increase  of  the 
typical  water  bacilli,  overbalanced  at  first  by  a  reduction 
in  other  forms,  for  which  this  is  an  unsuitable  environment. 

BACTERIAL  CHANGES   IN  WATER  DURING   STORAGE. 
(Whipple,  1901.) 


Temp. 

Number  of  Bacteria  per  c.c. 

Sample. 

Initial 
Temper- 

of Incu- 
bation 

ature. 

of 
Sample. 

Initial. 

After 
3  Hours. 

After 
6  Hours. 

After 
24  Hours. 

After 
48  Hours. 

C. 

C. 

A 

7.6° 

I7.o° 

260 

215 

230 

900 

27,000 

B 

7-6° 

I7.o° 

260 

245 

255 

720 

10  850 

C 

7-6° 

I2.5° 

260 

270 

231 

600 

2,790 

D 

7-6° 

12.5° 

260 

270 

245 

710 

1,  800 

E 

7.6° 

2.4° 

260 

243 

210 

675 

I  980 

F 

7.6° 

2.4° 

260 

235 

270 

560 

1,980 

G 

11.0° 

12.8° 

77 

55 

58 

101 

10.250 

H 

11.0° 

12.8° 

77 

53 

74 

87 

2'i75 

I 

11.0° 

23.6° 

77 

51 

52 

11,000 

41.400 

I 

6.7° 

6.7° 

20.0° 
20.0° 

43° 

4.2O 

375 

24  IT 

245 

4.CX 

385  ooo* 

7  £  O  OOO* 

L 

23.2° 

23-0° 

4-Ov 
5IO 

OT-J 
340 

t^o 
230 

8;ooo 

/  Jw<ww 

20,000 

M 

23.2° 

2-5° 

525 

300 

220 

380 

2;20O 

*  0.0005  per  cent  peptone  added  to  the  water. 

Wolffhugel  and  Riedel  (Wolffhugel  and  Riedel,  1886) 
noted  the  dependence  of  this  multiplication  on  the  amount 
of  the  air-supply,  vessels  closed  with  rubber  stoppers  show- 
ing lower  numbers  than  those  plugged  with  cotton.  Simi- 
larly, Whipple  found  that  the  multiplication  of  bacteria 


QUANTITATIVE  BACTERIOLOGICAL  EXAMINATION.   27 


was  much  greater  when  the  bottles  were  only  half  full 
than  when  they  were  filled  completely;  and  also,  as  shown 
in  the  following  very  striking  table,  that  the  size  of  the 
bottle  markedly  influenced  the  growth. 

EFFECT     OF    SIZE     OF    VESSEL     UPON     THE    MULTIPLICATION    OF    WATER 

BACTERIA   DURING   STORAGE. 

(Whipple,  1901.) 


Number  of  Bacteria  per  c.c. 

Temp,  of 

Sample 

Bottle. 

Incuba- 

tion. 

Ini- 

After 

After 

After 

After 

After 

tial.* 

3  hrs. 

6  hrs. 

12  hrs. 

24  hrs. 

48  hrs. 

C. 

A 

i  -gallon 

13° 

77 

63 

65 

47 

42 

175 

B 

2  -quart 

13° 

77 

59 

63 

60 

45 

690 

C 

i  -quart 

13° 

77 

63 

63 

47 

46 

325 

D 

i  -pint 

13° 

77 

57 

61 

36 

38 

630 

E 

2  -ounce 

13° 

77 

55 

58 

47 

IOI 

10,250 

F 

i  -gallon 

24° 

77 

81 

97 

275 

290 

300 

G 

2  -quart 

24° 

77 

92 

59 

62 

180 

250 

H 

i  -quart 

24°- 

77 

84 

77 

46 

340 

900 

I 

i  -pint 

24° 

77 

51 

46 

IOO 

2,950 

7,020 

J 

2  -ounce 

24° 

77 

51 

52 

145 

11,000 

41,400 

*  Average  of  five  plates. 

These  results  and  those  of  other  observers  make  it 
obvious  that  samples  must  be  examined  shortly  after 
collection  and  that  they  must  be  kept  cool  during  their 
storage.  If  fairly  pure  waters  are  placed  upon  ice  and  kept 
between  o°  and  10°,  they  will  show  no  material  increase 
in  twelve  hours.  With  polluted  water,  however,  another 
danger  is  here  introduced.  Samples  of  such  water  when 
packed  in  ice  show  a  marked  decrease  due  to  the  large 
number  of  sensitive  intestinal  bacteria  present.  Jordan 
(Jordan,  190x5)  found  that  three  samples  of  river- water 


28  ELEMENTS  OF   WATER  BACTERIOLOGY. 

packed  in  ice  for  forty-eight  hours  fell  off  from  535,000  to 
54,500;  from  412,000  to  50,500,  and  from  329,000  to 
73,000,  respectively.  It  is,  therefore,  necessary  to  adhere 
strictly  to  the  recommendations  of  the  A.  P.  H.  A.  Com- 
mittee that  the  interval  between  sampling  and  examina- 
tion should  not  exceed  twelve  hours  in  the  case  of  rela- 
tively pure  waters,  six  hours  in  the  case  of  relatively 
impure  waters,  and  one  hour  in  the  case  of  sewage. 

Plating. — The  bottle  containing  the  sample  of  water  is 
first  shaken  at  least  twenty-five  times  in  order  to  get  an 
equal  distribution  of  the  bacteria.  If  the  number  of 
bacteria  present  is  probably  not  greater  than  200,  i  c.c.  is 
then  withdrawn  with  a  sterile  i  c.c.  pipette  and  delivered 
into  a  sterile  Petri  dish  of  10  cm.  diameter.  To  this  is 
added  5  c.c.  of  standard  10  per  cent  gelatin  at  a  tempera- 
ture of  about  30°  C.  or  standard  agar  (7  c.c.)  at  4o°-42°  C 
Should  the  number  of  bacteria  per  c.c.  probably  exceed 
200,  dilution  is  necessary.  This  is  best  accomplished  by 
adding  i  c.c.  of  the  water  in  question  to  9,  99,  or  999,  etc., 
c.c.  of  sterile  tap  water  according  to  the  amount  of  dilution 
required.  The  diluted  sample  is  then  shaken  thoroughly 
and  i  c.c.  taken  for  enumeration.  In  order  to  determine 
the  number  of  bacteria  originally  present  it  is  only  neces- 
sary to  multiply  by  the  factor  10,  100,  or  1000,  etc. 

When  a  sample  of  water  from  an  unknown  source  is  to 
be  examined  it  is  generally  desirable  to  make  a  series  of 
plates  at  each  of  the  above  dilutions,  selecting  those  which 
give  nearest  to  200  colonies  on  the  plates  after  incubation 
as  the  ones  on  which  to  rely  for  the  count.  A  much 


QUANTITATIVE  BACTERIOLOGICAL  EXAMINATION.  29 

smaller  number  will  not  give  average  figures,  and  if  more 
than  200  colonies  are  present  on  a  plate  many  bacteria 
will  be  checked  by  the  waste  products  of  those  which  first 
develop  and  the  count  obtained  will  be  too  low.  After 
the  addition  of  the  diluted  sample  and  the  nutrient  medium 
their  thorough  mixture  in  an  even  layer  on  the  bottom 
of  the  plate  is  obtained  by  careful  tipping  and  rota- 
tion. 

It  was  formerly  customary  to  mix  the  water  with  the 
gelatin  in  the  tube  before  pouring  into  the  plate,  but  this 
method  is  objectionable  because  there  is  always  a  small 
residue  of  medium  remaining  in  the  tube  which  will 
retain  varying  numbers  of  bacteria  and  thus  interfere 
with  the  accuracy  of  the  count.  Before  pouring  the 
medium  into  the  plate  the  mouth  of  the  tube  should 
be  flamed  to  remove  any  possibility  of  contamina- 
tion. 

The  exact  composition  of  the  medium  used  is,  of  course, 
of  prime  importance  in  controlling  the  number  of  bac- 
teria which  will  develop.  The  reaction  of  the  medium 
was  found  as  early  as  1891  to  be  important,  for  Reinsch 
(Reinsch,  1891)  showed  in  that  year  that  the  addition 
of  one  one-hundredth  of  a  gram  of  sodium  carbonate  to 
the  liter  increased  sixfold  the  number  of  bacteria  develop- 
ing. Fuller  (Fuller,  1895)  and  Sedgwick  and  one  of 
ourselves  (Sedgwick  and  Prescott,  1895),  working  inde- 
pendently, established  the  fact  that  an  optimum  reac- 
tion existed  for  most  water  bacteria  and  that  a  deviation 
either  way  decreased  the  number  of  colonies  developing. 


30  ELEMENTS  OF   WATER  BACTERIOLOGY. 

The  following  table  from  Gage  and  Phelps  shows  con- 
clusively the  effect  of  the  general  composition  of  the 
nutrient  medium. 


TABLE  SHOWING  PERCENTAGES  OF  BACTERIA  DEVELOPING  ON  MEDIA  OF 

DIFFERENT   COMPOSITIONS. 

(Gage  and  Phelps,  1902.) 


• 

I 

)ays' 

Count 

2 

3 

4 

5 

6 

7 

8 

9 

Nahrstoff  agar 

in 

60 

78 

85 

otr 

00 

00 

IOO 

Nahrstoff  pepton  agar.  .  . 

10 

22 

26 

28 

3O 

3O 

3O 

3O 

Pepton  agar.  .... 

ii 

T6 

22 

23 

24 

24 

24 

24 

Meat  agar.    .          .    .        .... 

8 

13 

T6 

17 

I? 

17 

17 

17 

Plain  agar  

8 

IO 

13 

14 

14 

14 

14 

14 

Regular  agar  

7 

Q 

II 

II 

II 

II 

II 

II 

Nahrstoff  glycerin  agar  

6 

IO 

II 

II 

II 

II 

II 

II 

Nahrstoff  meat  agar  

7 

7 

8 

8 

IO 

IO 

IO 

IO 

Meat  gelatin  

12 

IO 

24 

06 

^6 

?6 

<?6 

26 

Pepton  gelatin 

7 

12 

18 

20 

20 

20 

20 

2O 

Standard  gelatin 

8 

IO 

1  1 

12 

I  3 

13 

13 

I  3 

Plain  gelatin 

i 

6 

12 

I  3 

13 

13 

13 

I  3 

Nahrstoff  gelatin  

c 

6 

Q 

II 

13 

13 

13 

13 

Finally,  Whipple  (Whipple,  1902)  has  shown  that  not 
only  the  particular  kind  of  gelatin  used  but  its  exact 
physical  condition  as  affected  by  sterilization  and  other 
previous  treatments  will  materially  affect  the  results 
obtained.  It  is  evident,  therefore,  that  only  the  strictest 
adherence  to  some  standard  method  can  ensure  com- 
parable results;  the  ordinary  nutrient  gelatin  should 
then  in  all  practical  sanitary  work  be  made  up  in  exact 
accordance  with  the  direction  of  the  A.  P.  H.  A.  Commit- 
tee as  given  in  the  Appendix. 


QUANTITATIVE  BACTERIOLOGICAL  EXAMINATION.  31 

Incubation. — "  Incubation  should  take  place  in  a  dark, 
well-ventilated  chamber  where  the  temperature  is  kept 
substantially  constant  at  20°  and  where  the  atmosphere 
is  practically  saturated  with  moisture." — A.  P.  H.  A. 
Report.  It  has  been  shown  by  Whipple  (Whipple,  1899) 
and  others  that  the  number  of  bacteria  developing  in 
plate  cultures  is  to  a  certain  extent  dependent  upon  the 
presence  of  abundant  oxygen  and  moisture.  Thus 
reckoning  the  number  of  bacteria  developing  in  a  moist 
chamber  at  100  the  percentage  counts  obtained  in  an 
ordinary  incubator  were  as  follows:  75  when  the  relative 
humidity  of  the  incubator  was  60  per  cent  of  saturation; 
82  when  it  was  75  per  cent;  98  when  it  was  95  per  cent. 
This  source  of  error  may  be  avoided  by  the  use  of  ven- 
tilated dishes  and  by  the  presence  of  a  pan  of  water  in 
the  incubating  chamber. 

According  to  American  and  German  practice,  plates 
made  for  sanitary  water  analysis  are  counted  at  the  end  of 
forty-eight  hours.  French  bacteriologists  still  recom- 
mend longer  periods,  and  the  following  table  from  Miquel 
and  Cambier  (Miquel  and  Cambier,  1902)  shows  that 
many  bacteria  fail  to  appear  in  our  ordinary  analysis. 
It  is,  however,  as  we  have  before  pointed  out,  certain 
peculiar  water  forms  which  develop  so  slowly,  sewage 
bacteria  almost  without  exception  being  rapid  growers. 
The  longer  period  of  incubation  is,  therefore,  not  only 
inconvenient  but  undesirable,  since  it  obscures  the  differ- 
ence between  good  and  bad  waters. 


32  ELEMENTS  OF  WATER  BACTERIOLOGY. 

EFFECT  OF  THE   LENGTH    OF   INCUBATION    OF    WATER   BACTERIA   IN   GEL- 
ATIN  UPON   THE   NUMBER   OF    COLONIES   DEVELOPING. 

(Miquel  and  Cambier,  1902.) 
Length  of  Incubation.  Colonies  Developed. 

1  day 20 

2  days 136 

3  "    254 

4  '    387 

5  "  530 

6  "  ..- 637 

7  "  725 

8  "  780 

9  "  821 

10  "    859 

11  "    892 

12  "      921 

13  "      951 

14  '    976 

15  "    1000 

Counting. — The  number  of  bacteria  is  determined  by 
counting  the  colonies  developed  upon  the  plate,  either 
with  the  naked  eye  or  preferably  with  a  low-power  lens. 
This  is  actually  done  by  placing  the  plate  upon  a  glass 
plate  ruled  in  centimeter  squares  and  over  a  black  tile; 
or  the  tile  itself  may  be  ruled.  The  colonies  then  appear 
as  whitish  round  or  oval  specks  in  and  upon  the  medium. 
As  has  already  been  said,  it  is  desirable  that  this  number 
should  not  exceed  200,  for  when  the  number  is  very  high 
the  colonies  grow  only  to  a  small  size  and  make  counting 
laborious  and  inaccurate,  and  many  do  not  develop  at  all. 
On  the  other  hand,  too  few  bacteria  upon  the  plate  prob- 
ably give  inaccurate  results  also.  The  greatest  accuracy 
is  probably  obtained  with  numbers  ranging  from  50  to 
200. 


QUANTITATIVE  BACTERIOLOGICAL  EXAMINATION.  33 


When  it  is  possible  to  do  so,  all  the  colonies  on  the 
plate  should  be  counted.  When  they  exceed  400  or  500 
it  is  often  easier  and  fully  as  accurate  to  count  a  frac- 
tional part  of  the  plate  and  estimate  the  total  number 
therefrom.  This  should  not  be  done,  however,  except 
in  case  of  necessity. 

It  is  customary  in  determining  numbers  to  make  plates 
in  duplicate,  thereby  affording  a  check  upon  one's  own 
work  and  enhancing  the  value  of  the  results  through 
the  greater  accuracy  obtained.  Owing  to  the  lack  of 
precision  in  the  method,  the  limit  of  experimental  error  is 
a  wide  one.  It  should  be  possible,,  however,,  for  careful 
manipulators  to  obtain  results  within  10  per  cent  of  eacfy 
other,  and  a  closer  agreement  than  this  is  hardly  to  be 
expected.  It  has  been  suggested  by  the  committee  of 
the  American  Public  Health  Association  to  adopt  the  fol- 
lowing mode  of  expressing  results. 

NUMBERS  OF    BACTERIA  FROM 

1-50  shall  be  recorded  to  the  nearest  unit 

51-100 

101-250 

251-500 

501-1,000 

1,001-10,000 

10,001-50,000 

50^001-100,000 

100,001-500,000 

500,001-1,000,000 

1,000,001-5,000,000 

The  determination  of  numbers  of  bacteria  in  water  in 
the  field  has  frequently  been  attempted.  Since  the  labora- 


1  " 

i      i 

5 

<  « 

«     < 

10 

<   « 

'   ' 

25 

(   « 

'     ' 

5° 

<   .« 

'     ' 

100 

(  ft 

'   ' 

500 

" 

' 

1,000 

10,000 

50,000 

1   (  I 

<   ' 

<«   t 

100,000 

34  ELEMENTS  OF  WATER  BACTERIOLOGY. 

tory  method  of  "plating  out"  is  not  applicable  for  field 
work  the  Esmarch  tube  process  has  often  been  employed. 
This  consists  in  introducing  into  a  tube  of  melted  gelatin 
or  agar  i  c.c.  of  the  water  and  then  rotating  the  tube  until 
the  medium  has  solidified  in  a  thin  layer  on  the  inner  wall 
of  the  tube.  In  the  writers'  opinion,  this  method  is  open 
to  several  objections  and  does  not  give  as  trustworthy  re- 
sults as  the  laboratory  method,  even  if  the  samples  have 
to  be  several  hours  in  transit  between  the  place  of  col- 
lection and  the  laboratory. 


CHAPTER  III. 


THE    INTERPRETATION    OF    THE    QUANTITATIVE    BAG- 
TERIOLOGICAL  ANALYSIS. 


THE  information  furnished  by  quantitative  bacteri- 
ology as  to  the  antecedents  of  a  water  is  in  the  nature  of 
circumstantial  evidence  and  requires  judicial  interpre- 
tation. No  absolute  standards  of  purity  can  be  estab- 
lished which  shall  rigidly  separate  the  good  from  the  bad. 
In  this  respect  the  terms  "test"  and  " analysis"  so  univer- 
sally used  are  in  a  sense  inappropriate.  Some  scientific 
problems  are  so  simple  that  they  can  be  definitely  settled 
by  a  test.  The  tensile  strength  of  a  given  steel  bar,  for 
example,  is  a  property  which  can  be  absolutely  deter- 
mined. In  sanitary  water  analysis,  however,  the  factors 
involved  are  so  complex  and  the  evidence  necessarily  so 
indirect  that  the  process  of  reasoning  much  more  re- 
sembles a  doctor's  diagnosis  than  an  engineering  test. 

The  older  experimenters  attempted  to  establish  arbi- 
trary standards,  by  which  the  sanitary  quality  of  a  water 
could  be  fixed  automatically  by  the  numbers  of  germs 
alone.  Thus  Miquel  (Miquel,  1891)  furnished  a  table 
according  to  which  water  with  less  than  10  bacteria  per  c.c. 
was  "excessively  pure,"  with  10  to  100  bacteria,  "very 

35 


36  ELEMENTS  OF   WATER  BACTERIOLOGY. 

pure,"  with  100  to  1000  bacteria,  "pure,"  with  1000  to 
10,000  bacteria,  "  mediocre,"  with  10,000  to  100,000  bac- 
teria, " impure,"  and  with  over  100,000  bacteria,  "very 
impure."  Few  sanitarians  would  care  to  dispute  the 
appropriateness  of  the  titles  applied  to  waters  of  the  last 
two  classes;  but  many  bacteriologists  have  placed  the 
standard  of  "purity"  much  lower.  The  limits  set  by 
various  German  observers  range,  for  example,  from  50  to 
.300.  Even  Dr.  Sternberg  (Sternberg,  1892),  in  a  much 
more  conservative  fashion,  has  stated  that  a  water  con- 
taining less  than  100  bacteria  is  presumably  from  a  deep 
source  and  uncontaminated  by  surface  drainage;  that 
one  with  500  bacteria  is  open  to  suspicion;  and  that  one 
with  over  1000  bacteria  is  presumably  contaminated  by 
sewage  or  surface  drainage.  This  is  probably  as  satisfac- 
tory an  arbitrary  standard  as  could  be  devised,  but  any 
such  standard  must  be  applied  with  great  caution.  The 
source  of  the  sample  is  of  vital  importance  in  the  inter- 
pretation of  analyses;  a  bacterial  count  which  would  con- 
demn a  spring  might  be  quite  normal  for  a  river;  only 
figures  in  excess  of  those  common  to  unpolluted  waters 
of  the  same  character  giye  the  indication  of  danger.  Fur- 
thermore, the  bacteriological  tests  are  far  more  delicate 
than  any  others  at  our  command,  very  minute  additions 
of  food  material  causing  an  immense  multiplication  of 
the  microscopic  flora.  This  delicacy  necessarily  requires, 
both  in  the  process  of  analysis  and  the  interpretation  of 
results,  a  high  degree  of  caution.  As  pointed  out  in  the 
previous  chapter,  the  touch  of  a  finger  or  a  particle  of 


QUANTITATIVE  BACTERIOLOGICAL  ANALYSIS.     37 

dust  may  wholly  destroy  the  accuracy  of  an  examination. 
Even  the  slight  disturbance  of  conditions  incident  upon 
the  storage  of  a  sample  after  it  has  been  taken  may  in  a 
few  hours  wholly  alter  the  relations  of  the  contained 
microbic  life.  It  is  necessary,  then,  in  the  first  place  to 
exercise  the  greatest  care  in  allowing  for  possible  error  in 
the  collection  and  the  handling  of  bacteriological  samples; 
and  in  the  second  place,  only  well-marked  differences  in 
numbers  should  be  considered  as  possibly  significant. 

In  the  early  days  of  the  science,  discussion  ran  high  as 
to  the  interpretation  of  bacteriological  analysis;  and  par- 
ticularly as  to  the  relation  of  bacterial  numbers  to  the 
organic  matter  present  in  a  water.  Different  observers 
obtained  inconsistent  results,  and  Bolton  (Bolton,  1886) 
concluded  that  there  was  no  relation  whatever  between 
the  chemical  composition  of  a  water  and  its  bacterial  con- 
tent. Tiemann  and  Gartner  (Tiemann  and  Gartner, 
1889)  furnished  the  key  to  the  difficulty  in  their  state- 
ment that  there  are  two  great  classes  of  bacteria,  the 
great  majority  of  species  normally  occurring  in  the  earth 
or  in  decomposing  organic  matter  which  require  abun- 
dance of  nutriment,  and  certain  peculiar  water  bacteria 
which  can  multiply  in  the  presence  of  such  minute  traces 
of  ammonia  as  are  present  in  ordinary  distilled  water. 
Bacteria  of  the  second  class  under  abnormal  conditions, 
as  in  bottled  samples,  or,  at  times,  in  a  well  or  the  basin  of 
a  spring,  may  occur  in  great  numbers  where  there  is  but 
little  organic  matter.  In  normal  surface-waters,  how- 
ever, such  growths  have  not  been  recorded  as  far  as  we 


38  ELEMENTS  OF  WATER  BACTERIOLOGY. 

are  aware.  Here  the  numbers  of  bacteria  present  will 
depend,  other  things  being  equal,  upon  the  extent  to 
which  he  water  has  been  contaminated  with  decomposing 
organic  matter,  either  by  pollution  with  sewage  o:  by 
contact  with  the  surface  of  the  ground.  The  bacterial  con- 
tent will  therefore  vary  as  the  extent  and  character  of  the 
contamination  varies  It  measures  not  merely  organic 
matter  but  organic  matter  in  a  state  of  active  decay,  and 
thus,  like  the  ammonias  and  other  features  of  the  sanitary 
chemical  analysis,  clearly  indicate,  fresh  organic  pollution 
with  the  added  advantage  that  the  presence  of  the  stable 
nitrogenous  compounds  often  present  in  peaty  waters 
introduces  no  error  in  the  bacteriological  analysis. 

In  judging  of  a  surface-water  the  student  will  be  aided 
by  reference  to  the  figures  given  for  certain  normal  sources 
in  Chapter  I;  the  Boston  tap  water  with  50  to  200  bac- 
teria per  c.c.  (Whipple,  1896)  and  the  water  of  Lake 
Zurich  with  an  average  of  71  in  summer  and  184  in  winter 
(Cramer,  1885)  may  be  taken  as  typical  of  good  potable 
waters;  and  numbers  much  higher  than  these  are  open 
to  suspicion,  since  all  contamination  whether  contributed 
by  sewage  or  by  washings  from  the  surface  of  the  ground 
is  a  possible  source  of  danger.  The  excess  of  bacteria 
in  surface-waters  during  the  spring  and  winter  months  is 
by  no  means  an  exception  to  the  general  rule  that  high 
numbers  are  significant,  since  the  peril  from  supplies  of 
this  character  is  clearly  shown  by  the  spring  epidemics  of 
typhoid  fever  which  at  the  times  of  melting  snow  visit 
communities  making  use  of  unprotected  surface-waters. 


QUANTITATIVE  BACTERIOLOGICAL  ANALYSIS.     39 

Streams  receiving  direct  sewage  pollution  exhibit  a  simi- 
lar excess  of  bacteria  at  all  times,  numbers  rising  to  an 
extraordinary  height  near  the  point  of  entrance  and  fall- 
ing off  below  as  the  stream  suffers  dilution  and  the  sewage 
organisms  perish.  Miquel  (Miquel,  1886)  records  300 
bacteria  per  c.c.  in  the  water  of  the  Seine  at  Choisy,  above 
Paris,  1200  at  Bercy  in  the  vicinity  of  the  city,  and  200,000 
at  St.  Denis  after  the  entrance  of  the  drainage  of  the  city. 
Prausnitz  (Prausnitz,  1890)  found  531  bacteria  per  c.c. 
in  the  Isar  above  Munich,  227,369  near  the  entrance  of 
the  principal  sewer,  9111  a  a  place  13  kilometers  below 
the  city,  and  2378  at  Freising,  20  kilometers  further 
down.  Jordan  (Jordan,  1900),  in  his  study  of  the  fate  of 
the  sewage  of  Chicago,  found  1,245,000  bacteria  per  cc. 
in  the  drainage  canal  at  Bridgeport,  650,000  twenty-nine 
miles  below  at  Lockport,  and  numbers  steadily  decreasing 
below  to  3660  at  Averyville,  159  miles  below  the  point  of 
original  pollution.  Below  Averyville  the  sewage  of  Peoria 
enters  and  the  numbers  rise  to  758,000  at  Wesley  City, 
decreasing  to  4800  in  123  miles  flow  to  KampsviUe. 

In  ground-waters  we  have  seen  that  bacteria  may  be 
present  in  considerable  numbers,  but  that  they  will 
generally  be  organisms  of  a  peculiar  character,  incapable 
of  development  on  the  ordinary  nutrient  media  in  the 
standard  time.  Thus  in  forty-eight  hours  we  often  obtain 
counts  measured  only  in  units  or  tens.  When  higher 
numbers  are  present,  the  general  character  of  the  colonies 
must  be  taken  into  account,  since  besides  the  slowly- 
growing  forms  certain  other  water  bacteria  which 


40  ELEMENTS  OF  WATER  BACTERIOLOGY. 

require  a  comparatively  small  amount  of  nutriment  may 
multiply  at  times  in  a  deep  well  or  the  basin  of  a 
spring.  In  such  a  case,  however,  the  appearance  of  the 
plates  would  at  once  reveal  the  peculiar  conditions,  for  the 
colonies  would  all  be  of  one  kind  and  that  distinct  from 
any  of  the  sewage  species.  Thus  Dunham  (Dunham,  1889) 
reports  that  the  mixed  water  from  a  series  of  driven  wells 
gave  2  bacteria  per  c.c.,  while  another  well,  situated  just 
like  the  others,  contained  5000,  all  belonging  to  a  single 
species  common  in  the  air.  Plates  from  polluted  water 
contain,  on  the  other  hand,  a  wide  variety  of  species. 

The  process  of  slow  sand  nitration  for  the  purification 
of  unprotected  surface-water  is  essentially  similar  to  the 
action  which  takes  place  in  nature  when  rain  soaks  through 
the  ground  to  appear  in  wells  and  springs;  and  it  is  in  the 
examination  of  the  effluent  from  such  municipal  plants 
that  the  quantitative  bacteriological  analysis  finds,  per- 
haps, its  most  important  application.  The  chemical 
changes  which  occur  in  the  passage  of  water  through 
sand  at  a  rate  of  1,000,000  or  2,000,000  gallons  per  acre 
per  day  are  so  slight  as  to  be  negligible.  The  bacteria 
present  should,  however,  suffer  a  reduction  of  98  or  99  per 
cent,  and  their  numbers  are,  therefore,  used  as  the  standard 
for  measuring  the  efficiency  of  such  filtration  plants.  At 
Lawrence,  in  1901,  Clark  found  an  average  of  3017  bac- 
teria per  cc.  in  the  raw  water  of  the  Merrimac  River,  while 
the  number  present  in  the  filtered  water  was  only  26 
(Massachusetts  State  Board  of  Health,  1902).  Mechan- 
ical filtration  gives  similar  results.  Fuller  at  Cincinnati 


QUANTITATIVE  BACTERIOLOGICAL  ANALYSIS.     41 

(Fuller,  1899)  records  27,200  organisms  per  c.c.  in  the 
water  of  the  Ohio  River  between  September  21,  1898,  and 
January  25, 1899,  while  the  average  content  of  the  effluent 
from  the  Jewell  filter  was  400.  In  well-managed  purifi- 
cation plants  the  bacteria  in  the  effluent  are  determined 
daily,  and  any  deviation  from  the  normal  value  at  once 
reveals  disturbing  factors  which  may  impair  the  efficiency 
of  the  process.  In  Prussia  official  regulations  demand 
such  systematic  examinations  and  prescribe  50  as  the 
maximum  number  of  bacteria  allowable  in  the  filtered 
water.  In  the  same  way  the  condition  of  an  unpurified 
surface  supply  may  be  determined  by  daily  bacteriological 
analyses  and  warnings  of  danger  issued  to  the  public,  as 
has  been  done  at  Chicago  and  other  cities.  In  general, 
any  such  regular  determination  of  variations  from  a  nor- 
mal standard  furnish  ideal  conditions  for  the  bacteriolog- 
ical methods;  and  the  detection  by  Shuttle  worth  (Shuttle- 
worth,  1895)  of  a  break  in  a  conduit  under  Lake  Ontario 
by  a  rise  in  the  bacteria  of  the  Toronto  water-supply  may 
be  cited  as  a  classic  example  of  its  application. 

Often,  however,  the  expert  is  called  to  pass  upon  the  char- 
acter of  a  water  of  which  no  series  of  analyses  is  available 
and  whose  surroundings  it  may  be  impossible  for  him  to  in- 
spect. It  has  been  said  that  single  bacteriological  analyses 
of  this  kind  are  valueless;  but  this  we  believe  cannot  always 
be  maintained.  Knowing  the  normal  bacterial  range 
for  a  given  class  of  waters,  even  an  isolated  analysis  may 
show  such  an  excess  as  to  have  great  significance,  as  a 
few  practical  examples  may  make  clear  (Winslow,  1901). 


42  ELEMENTS  OF  WATER  BACTERIOLOGY. 

In  the  spring  of  1900  the  city  of  Hartford,  Conn.,  was 
using  a  double  supply  from  the  Connecticut  River  and 
from  a  series  of  impounding  reservoirs  among  the  hills. 
A  single  series  of  plates  showed  from  4000  to  7000  bac- 
teria per  c.c.  in  the  water  of  the  river,  while  the  reservoir 
water  contained  300  to  900.  The  abandonment  of  the 
river  supply  followed,  and  at  once  the  excessive  amount 
of  typhoid  fever  in  the  city  was  curtailed. 

In  the  fall  of  1900,  Newport,  R.  L,  experienced  an  out- 
break of  typhoid  fever,  and  when  suspicion  was  thrown 
upon  the  public  water-supply,  chemical  analysis  of  the 
latter  was  not  wholly  reassuring;  but  there  were  only 
334  bacteria  per  c.c.  in  the  water  from  the  taps,  while  a 
well  in  the  infected  district  gave  6100.  It  was  no  surprise 
to  find,  on  a  further  study  of  the  epidemic,  that  the  well 
was  largely  at  fault  and  the  public  supply  not  at  all. 

In  the  case  of  ground- water  the  evidence  is  usually  even 
more  distinct.  At  Framingham,  Mass.,  in  1903,  high 
chlorin  content  in  the  public  supply,  drawn  from  a  filter 
gallery  beside  a  lake,  had  led  to  public  anxiety.  Five 
samples  from  different  parts  of  the  system  showed  aver- 
ages of  i,  2,  2,  2,  and  4  bacteria  per  c.c.;  and  taking  this 
in  conjunction  with  the  other  features  of  the  bacterio- 
logical analysis,  it  was  possible  to  report  that  any  pollution 
introduced  upon  the  gathering  ground  had  at  the  time  of 
analysis  been  entirely  removed.  In  such  a  case  the  bac- 
teriological methods  give  a  certainty  attainable  by  no 
other  sanitary  process. 


CHAPTER  IV. 

DETERMINATION    OF    THE    NUMBER     OF    ORGANISMS 
DEVELOPING  AT  THE  BODY  TEMPERATURE. 

THE  count  of  colonies  upon  the  gelatin  plate  measures 
as  we  have  pointed  out,  the  number  of  those  micro-organ- 
isms associated  with  the  decomposition  of  organic  matter 
wherever  it  may  occur.  In  this  great  class,  however, 
there  are  a  few  species,  like  B.  subtilis  and  B.  ramosus, 
which  will  grow  under  a  great  variety  of  conditions  and 
which  are  likely  to  be  present  with  more  or  less  constancy 
in  water,  and  others  which  through  a  semi-parasitic 
mode  of  life  have  become  specially  adapted  to  the  pecu- 
liar conditions  characteristic  of  the  animal  body.  The 
latter  in  particular  possess  the  property  of  developing 
most  actively  at  the  temperature  of  the  human  organism, 
37°  C.,  which  altogether  checks  the  growth  of  the  majority 
of  normal  earth  and  water  forms.  The  determination  of 
the  number  of  organisms  growing  at  the  body  temperature 
may  throw  light,  then,  on  the  presence  of  direct  sewage 
pollution,  since  the  bacteria  from  the  alimentary  canal 
flourish  under  such  conditions,  while  most  of  those  derived 
from  other  sources  do  not.  The  count  at  37°  helps  to 
distinguish  contamination  by  wash  of  the  soil  of  a  virgin 
woodland  from  pollution  by  excreta,  since  in  the  latter 

43 


44  ELEMENTS  OF   WATER   BACTERIOLOGY. 

case  the  proportion  of  blood-temperature  organisms  is 
much  smaller  than  in  the  latter.  Furthermore,  this 
method  is  free  from  much  of  the  error  introduced  by  the 
multiplication  of  bacteria  after  the  collection  of  a  sample, 
since  most  of  the  forms  which  grow  in  water  during 
storage  cannot  endure  the  higher  temperature  and  conse- 
quently do  not  develop  upon  incubation. 

The  body- temperature  count  must,  of  course,  be  made 
upon  agar  plates,  otherwise  the  technique  is  the  same 
which  has  already  been  described  for  the  routine  quan- 
titative bacteriological  analysis.  The  period  of  incuba- 
tion ordinarily  adopted  by  the  writers  is  twenty-four 
hours,  as  little  development  occurs  after  that  time.  Diffi- 
culty is  sometimes  caused  by  the  spreading  of  colonies 
of  certain  organisms  over  'the  surface  of  the  plate  in  the 
water  of  condensation  which  gathers;  this  may  be  avoided 
by  inverting  the  plates  after  the  agar  is  once  well  set. 

Additional  evidence  as  to  the  character  of  a  water 
sample  may  be  obtained  with  little  extra  labor  by  adding 
a  sugar  and  some  sterile  litmus  to  the  agar  medium  and 
observing  the  fermenting  powers  of  the  organisms  present, 
as  first  suggested  by  Wurtz  (Wurtz,  1892)  for  the  separa- 
tion of  B.  coli  from  B.  typhi.  It  happens  that  the  most 
abundant  intestinal  organisms  belonging  to  the  groups 
of  the  colon  bacilli  and  the  streptococci  decompose  dex- 
trose and  lactose  with  the  formation  of  a  large  excess  of 
acid,  while  many  other  organisms,  even  if  they  grow 
abundantly  at  the  body  temperature,  are  not  favored  by 
the  presence  of  the  sugar  and  litmus.  The  decomposi- 


DETERMINATION  OF  ORGANISMS.  45 

tion  of  the  latter  sugar  is  almost  entirely  wanting  among 
the  commoner  saprophytic  bacteria,  and  therefore  lac- 
tose is  most  commonly  used  in  making  sugar  agar,  2  per 
cent  being  added  to  the  medium  just  before  the  final  nitra- 
tion (between  steps  15  and  16  in  the  standard  process 
of  media  making  given  on  p.  120).  In  pouring  the  plate 
a  cubic  centimeter  of  sterile  litmus  solution  should  be 
added;  and  in  counting,  the  colonies  of  the  acid-forming 
organisms  will  be  clearly  picked  out  by  the  reddening  of 
the  adjacent  agar.  Only  those  which  show  this  clearly 
should  be  considered  as  significant,  since  certain  bacteria 
of  the  hay-bacillus  group  produce  weak  acid  and  faint 
coloring  of  the  litmus. 

When  polluted  waters  are  examined  in  this  manner  the 
number  of  organisms  developing  on  the  lactose-agar  plate 
will  be  very  high,  almost  equalling  in  some  cases  the  total 
count  obtained  on  gelatin.  Chick  (Chick,  1901),  using 
a  lactose-agar  medium  with  the  addition  of  one-thousandth 
part  of  phenol,  found  of  colon  bacilli  alone  6100  per  c.c. 
in  the  Manchester  ship  canal,  55-190  in  the  polluted 
River  Severn,  and  numbers  up  to  65,000  per  gram  in  road- 
side mud.  In  an  examination  of  water  from  the  Charles 
River  above  Boston,  total  37°  counts  ranging  from  9800  to 
16,900  have  been  found.  Two  twenty-four-hour  exam- 
inations of  Boston  sewage  made  at  the  Sanitary  Research 
Laboratory  of  the  Institute  of  Technology  during  the 
summer  of  1903  gave  an  average  of  3,660,000  bacteria 
per  c.c.  on  gelatin  at  20°  and  2,310,000  per  c.c.  on  lactose 
agar  at  37°  with  550,000  acid-formers. 


46 


ELEMENTS  OF   WATER  BACTERIOLOGY. 


In  unpolluted  waters  not  only  the  absolute  number  of 
organisms  developing  at  the  body  temperature,  but  its 
ratio  to  the  gelatin  count,  is  very  different.  Rideal  (Rideal, 
1902)  states  that  the  proportion  between  the  two  counts 
in  the  case  of  a  London  water  in  a  year's  examination 
was  on  the  average  one  to  twelve.  Mathews  (Mathews, 
1893)  in  1893,  gave  the  following  figures,  the  contrast 
between  the  ponds  and  streams  which  were  presumably 
exposed  to  pollution  on  the  one  hand,  and  the  wells, 
springs,  and  taps  on  the  other,  being  marked. 


Sources  of  Water. 

Average  Number  of 
Colonies  per  c.c. 

Gelatin,  20°. 

Wurtz  Agar, 
37-5°. 

^Vells  springs                               

1664 

*53 
296 
242 
273 

28 
43 
95 
24 

101 

Reservoirs                                   

Ponds                                     

Taps                                   

In  1903  Nibecker  and  one  of  ourselves  (Winslow  and 
Nibecker,  1903)  made  an  examination  of  259  samples 
of  water  from  presumably  unpolluted  sources  in  Eastern 
Massachusetts,  including  public  supplies,  brooks,  springs, 
ponds,  driven  wells,  and  pools  in  the  fields  and  woods, 
with  a  view  to  testing  the  value  of  the  body- temperature 
examination.  In  many  cases  the  samples  showed  high 
gelatin  counts,  since  some  of  the  waters  were  exposed  to 
surface  wash  from  vacant  land,  but  the  average  number 
of  organisms  developing  on  lactose  agar  at  37°  was  less 


DETERMINATION  OF  ORGANISMS. 


47 


RELATION  OF  2O°  AND  37°  COUNTS  IN  SAMPLES   OF  WATER    FROM    APPAR- 
ENTLY  UNPOLLUTED   SOURCES. 
(Winslow  and  Nibecker,  1903.) 


Source  of  Samples. 

Number  of  Samples. 

jelatin 
Plates, 

20°. 

Litmus- 
lac  tose- 
agar 
Plates,  37°. 

Dextrose  Broth 
Tubes. 

Average  Number 
of  Colonies. 

1  Average  Number 
of  Colonies. 

1  Plates  Showing 
Red  Colonies. 

Number  of  Tubes. 

1 

H 

!J 

1- 

1* 

It 

11 

Zz 

|'0 

Number  of  Tubes 
with  Gas  a-i. 

Cambridge  supply  (tap)  
Wakefield  and  Stoneham  supply 
(tan)                                 

5 

6 
i 

94 

59 
16 

II 
6 
18 

2 
21 

I 

9 
14 

2 
46 

8 
o 

12 

7 

I 

2 

9 
4 
3i 
3 
4 
o 
o 

2 

1 

2 

I 

O 

O 
0 
2 
0 
0 
0 
0 
O 

o 

2 
O 
O 
O 
O 
O 
O 
0 
0 
0 

o 
o 

0 

o 
o 
o 
o 

0 
0 

15 
21 

18 

18 

9 

18 
18 
15 

12 

9 

15 
6 

3 

183 
45 
95 
45 
3 
66 

65 
3° 
3 

18 
3 

9 

21 

775 

0 

0 
0 
0 

o 

2 
O 
O 

o 
o 

3 
o 

0 

o 

13 

o 

13 

I 

0 
2 
O 
2 
0 
O 
O 
0 

5 

o 
o 

41 

O 

0 

o 
o 
o 

2 
O 
O 
O 
O 
O 
O 
O 

o 

13 

o 

13 

I 
o 

2 
O 
2 

O 

0 

o 

0 

5 

o 
o 

38 

o 

0 

o 
o 

0 
0 
0 

o 
o 
o 

3 
o 
o 
-o 
o 
o 
o 

0 
0 
0 
0 

o 
o 
o 
o 
o 
o 

0 
0 

6 

3 
6 
6 

5 
4 
3 
5 

2 

I 

61 

15 
32 

15 

i 

35 
141 

3>7!7 
36 
232 

£ 

524 
4,700 

Newburyport  supply  (tap)  

Westerly,  R.  I.,  supply  (tap).  .  . 
Brooks  

223 

18 

294 
167 

Ponds  fed  by  brooks                .    - 

Jvlelted  snow                           .... 

Pools  in  fields  ...           ....... 

22 
22 
IO 

I 
2 

6 

i 

* 

365 
181 
811 

47 
1  88 
1,235 
269 

Pools  in  woods       ............ 

Stream,  Blue  Hill  Reservation.  . 
Flow  from  rocks 

Ponds  fed  by  springs 

Drainage  from  manured  pasture. 
^\vamps 

Rain-water  after  twelve   hours' 
heavy  fall 

Shallow  well  in  Lynn  woods.  .  .  . 

I 

15 

Totals  

259 

4 

3 

48  ELEMENTS  OF  WATER  BACTERIOLOGY. 

than  8  per  c.c.  The  highest  individual  counts  obtained, 
as  will  be  seen  by  reference  to  the  table  on  the  preceding 
page,  were  95  in  a  meadow  pool,  83  in  a  brook,  and  74 
in  a  barnyard  well,  the  latter  probably  actually  polluted. 
Only  two  samples  in  the  whole  series,  one  from  the  well 
above  mentioned,  gave  any  red  colonies  on  the  agar 
plates. 

Thus  it  is  clear  that  organisms  growing  at  the  body  tem- 
perature and  those  fermenting  lactose  are  not  numerous 
in  normal  waters,  the  total  count  rarely  exceeding  50, 
with  acid  producers  generally  entirely  absent.  On  the 
other  hand,  the  numbers  on  the  litmus-lactose-agar  plate 
will  be  likely  to  run  into  hundreds  with  a  good  proportion 
of  red  colonies  when  polluted  waters  are  examined.  The 
method  is,  therefore,  one  of  the  most  useful  at  the  dis- 
posal of  the  bacteriologist.  It  yields  results  within 
twenty-four  hours,  and  the  conclusions  to  be  drawn  from 
it  are  definite  and  clear. 


CHAPTER  V. 

THE  ISOLATION  OF  SPECIFIC  PATHOGENES  FROM 
WATER. 

THE  discovery  of  the  organisms  which  specifically 
cause  the  infectious  diseases  naturally  led  to  the  hope 
that  their  isolation  from  polluted  water  might  become 
the  most  convincing  proof  of  its  sanitary  quality.  The 
typhoid  bacillus  and  the  spirillum  of  Asiatic  cholera  were 
in  this  connection  of  paramount  importance,  and  to  the 
search  for  them  many  investigators  devoted  themselves. 

In  the  earlier  examinations  of  water  for  the  typhoid 
bacillus  an  attempt  was  made  to  use  media  which  espe- 
cially favored  the  growth  of  the  microbe  sought  for,  or  to 
begin  with  some  process  of  "  enrichment "  in  which  the 
sample  was  incubated  under  conditions  which  would 
favor  the  growth  of  the  pathogenic  organisms  while 
checking  the  development  of  the  common  water  bacteria. 
It  was  apparent  that  the  body  temperature  and  the  pres- 
ence of  a  slight  excess  of  free  acid  furnished  such  condi- 
tions, and  most  of  the  methods  suggested  rest  upon 
these  principles.  The  most  general  perhaps  is  that  of 
Parietti  (Parietti,  1890),  which  consists  in  the  addition 
of  the  water  to  a  series  of  broth  tubes  containing  increas- 

49 


50  ELEMENTS  OF  WATER  BACTERIOLOGY. 

ing  amounts  of  a  solution  of  4  per  cent  hydrochloric  acid 
and  5  per  cent  phenol.  From  tubes  in  which  growth 
occurs  after  twenty- four  hours  at  37°,  the  organisms 
present  may  be  isolated  in  pure  cultures  by  some  plating 
method  and  identified  by  subcultures. 

The  great  difficulty  with  the  enrichment  processes  is 
that  the  conditions  which  favor  the  multiplication  of  the 
typhoid  bacillus  are  suited  in  an  even  higher  degree  to 
B.  coli  and  other  intestinal  organisms.  Being  present  in 
almost  all  cases  in  much  higher  numbers  than  B.  typhi, 
these  germs  develop  most  abundantly,  and  effectually 
mask  any  disease  germs  originally  present.  In  order  to 
obviate  this  difficulty,  Hankin  (Hankin,  1899),  after 
adding  successively  increasing  portions  of  Parietti  solu- 
tion to  tubes  inoculated  with  the  water  to  be  tested, 
selected  the  second  highest  tube  of  the  series  in  which 
growth  occurs  for  the  inoculation  of  a  new  set,  finally 
plating  as  above.  He  believed  that  the  chance  for  over- 
growth in  this  method  is  somewhat  decreased;  but  in 
the  hands  of  other  investigators  it  has  not  met  with  marked 
success.  Klein  (Thomson,  1894),  in  his  investigations, 
made  use  of  the  Berkefeld  filter  to  concentrate  the  or- 
ganisms in  the  sample.  Some  recent  observers  have 
abandoned  the  enrichment  process  altogether  and  recom- 
mend direct  plating  upon  phenolated  gelatin  or  on  the 
Eisner  (Eisner,  1896)  medium  made  by  adding  10  per 
cent  of  gelatin  and  i  per  cent  of  potassium  iodide  to  an 
infusion  of  potato  whose  reaction  has  been  adjusted  to 
30  on  Fuller's  scale.  In  all  cases,  however,  the  chance 


ISOLATION  OF  SPECIFIC  PATHOGEN ES.  51 

of  success  is  small;  as  is  well  shown  by  the  experiments 
of  Laws  and  Andre wes  (Laws  and  Andre wes,  1894),  who 
entirely  failed  to  isolate  the  typhoid  bacillus  from  the 
sewage  of  London  and  found  only  two  colonies  of  the 
organism  on  a  long  series  of  plates  made  from  the  sewage 
of  a  hospital  containing  forty  typhoid  patients.  So  Wathe- 
let  (Wathelet,  1895)  found  that  of  600  colonies  isolated 
from  typhoid  stools  and  having  the  appearance  charac- 
teristic of  B.  coli  and  B.  typhi  only  10  belonged  to  the 
latter  species. 

At  the  other  end  of  the  process  the  identification  of  the 
pure  cultures-  isolated  is  again  subject  to  considerable 
uncertainty.  The  typhoid  bacillus  belongs  to  a  large 
group  which  contains  numerous  varieties  differing  from 
each  other  by  minute  degrees.  The  inability  to  repro- 
duce the  disease  by  inoculation  in  available  test  animals 
owing  to  their  natural  immunity  is  a  serious  drawback; 
and  the  specific  biochemical  characters  of  the  organism 
are,  as  it  happens,  mostly  negative  ones,  as  shown  by  com- 
parison with  B.  coli,  to  which  it  is  supposed  to  be  allied. 


COMPARISON   OF   THE   CHARACTERS   OF   B.    COLI   AND   B.   TYPHI. 

(Horrocks,  1901.) 
C.  coli.  B.  typhi. 

(i)  Surface    Colonies,     Gelatin        (i)  Much    thinner   than    those 

Plates. — Thicker,  and  grow  more  of  B.  coli,  and  grow  more  slowly, 

rapidly   than   those   of    B.   typhi.  After    forty-eight    hours'  incuba- 

After  forty-eight  hours'  incubation  tion   at   22°   C.   they   are   hardly 

at  22°  C.  they  are  usually  large  visible  to  the  naked  eye. 
and  characteristic. 


ELEMENTS  OF   WATER  BACTERIOLOGY. 


C.  coli. 

(2)  Gelatin-stab. — Quick  growth 
on  the  surface  and  along  the  line 
of  inoculation. 


(3)  Gelatin-slope. — Thick  broad 
grayish-white  growth  with   a  cre- 
nated  margin. 

(4)  Witte's   Peptone    and    Salt 
Solution. — Indol  produced. 

(5)  Milk.— Coagulated. 

(6)  Litmus-whey,  one  week  at 
37°    C.     Acid    produced,    usually 
requiring  from  20  to  40  per  cent  of 

N 

—  alkali  to  neutralize  it. 

10 

(7)  Neutral-red  Glucose-agar. — 
Marked  green  fluorescence. 

(8)  Glucose-gelatin     and     Lac- 
tose-gelatin   Shake   Cultures,    and 
Glucose-agar-stab. — Marked     gas 
formation. 

(9)  Gelatin,  25  per  cent,    incu- 
bated    at     37°     C.— Thick     film 
appears  on  the  surface. 

(10)  Potato. — As  a  rule,  a  thick 
yellowish-brown  growth. 

(n)  Proskauer  and  Capaldi's 
Media.  No.  I,  after  twenty  hours 
growth,  medium  acid.  No.  II, 
Growth,  medium  neutral  or  faintly 
alkaline. 

(12)  Nitrate-broth. — Nitrate  re- 
duced to  nitrite. 

(13)  Microscopical       Appear- 
ances.— A  small  bacillus  often  like 
a  coccus,  not  motile  as  a  rule. 

(14)  Flagella. — Usually  i  to    3, 
short  and  brittle;   sometimes  8  to 
12,  long  and  wavy. 


B.  typhi. 

(2)  Slow  growth  on  the  surface 
like  the  colonies;   along  the  line  of 
inoculation    the    growth   is   much 
thinner,  and  often  ends  below  in  a 
few  white  points  consisting  of  dis- 
crete colonies. 

(3)  Thin  narrow  grayish-white 
growth,      crenated      margin     not 
marked  as  in  B.  coli. 

(4)  No  formation  of  indol. 

(5)  Unchanged  after  a  month. 

(6)  Very  small  amount  of  acid 
produced,  requiring  not  more  than 

N 

6  per  cent  of  —  alkali  to  neutralize 
10 

it. 

(7)  No  change. 

(8)  No  gas  formation. 


(9)  No  film  appears  on  the  sur- 
face, but  a  general  growth  takes 
place  throughout  the  tube. 

(10)  Thin    transparent    growth 
hardly  visible  to  the  naked  eye. 

(n)  No.  I,  no  growth  or  change 
in  the  reaction  of  the  medium. 
No.  II,  Growth,  medium  acid. 


(12)  Reduction  of  nitrate  not  so 
marked. 

(13)  Usually    longer    than     B. 
coli;    highly  motile,  with  a  quick 
serpent-like  movement. 

(14)  Usually  8  to  12,  long  and 
wavy. 


ISOLATION  OF  SPECIFIC  PATHOGEN ES.  53 

(15)  Agglutination. — As  a  rule,         (15)  Marked  agglutination  with 
no  agglutination  with  a  dilute  anti-     dilute  anti-typhoid  serum, 
typhoid  serum. 

Of  the  many  observers  who  have  reported  the  isola- 
tion of  the  typhoid  bacillus  from  water  all  but  the  most 
recent  are  quite  discredited,  on  account  of  the  insufficiency 
of  their  confirmatory  tests,  and  even  the  latest  results 
should  be  received  with  caution.  Since  the  introduction 
of  the  Widal  (Widal,  1896)  reaction,  founded  on  the 
fact  that  typhoid  bacilli  examined  under  the  microscope 
in  the  diluted  blood-serum  of  a  typhoid  patient  lose  their 
motility  and  "  agglutinate "  or  clump  together,  an  import- 
ant aid  has  been  furnished  in  the  diagnosis.  Yet  serum 
tests  are  notably  erratic,  and  insufficient  to  identify  an 
organism  without  an  exhaustive  study  of  biochemical 
reactions,  especially  as  many  organisms  are  agglutinated 
by  typhoid  serum  in  a  more  or  less  dilute  solution.  The 
discovery  of  the  Bacillus  dysenteriae  of  Shiga,*  which 
closely  resembles  the  typhoid  bacillus,  has  made  the 
identification  of  the  latter  more  dubious  than  ever. 

It  seems  probable  that  in  some  recent  cases  the  typhoid 
bacillus  has  indeed  actually  been  isolated  from  polluted 
water,  as  by  Kiibler  and  Neufeld  (Klibler  and  Neufeld, 
1899),  who  examined  a  farmhouse  well  at  Neumark  in 
1899,  and  Fischer  and  Flatau  (Fischer  and  Flatau,  1901), 
who  discovered  an  organism  responding  to  a  most  ex- 
haustive series  of  tests  for  the  typhoid  bacillus  in  a  well  at 


*  For  an  account  of  the  Biology  of  B.  dysenteriae  the  student  is 
referred  to  an  article  by  Dombrowsky,  1903. 


54  ELEMENTS   OF  WATER  BACTERIOLOGY. 

Rellmgen  in  1901.  In  these  cases  the  water  was  directly 
plated  upon  Eisner's  medium  or  phenolated  gelatin  with 
no  preliminary  process  of  enrichment. 

The  search  for  the  typhoid  bacillus  is  usually  suggested 
when  an  outbreak  of  the  disease  has  cast  strong  suspicion 
upon  some  definite  source  of  water-supply.  By  the  time 
an  epidemic  manifests  itself,  however,  the  period  of  the 
original  infection  is  long  past,  and  the  chances  are  good 
that  any  of  the  specific  bacilli  once  present  will  have  dis- 
appeared. While  elaborate  experiments  have  shown 
that  B.  typhi  may  persist  in  sterilized  water  for  upwards 
of  two  months  and  in  unsterilized  water  from  three  days 
to  several  weeks,  the  number  of  the  organisms  present  is 
always  very  rapidly  reduced  (Frankland,  1894).  Epi- 
demiological  evidence  confirms  the  results  of  Laws  and 
Andre wes  which  teach  that  the  number  of  typhoid  bacilli 
even  in  polluted  water  probably  is  never  very  great,  while 
the  fate  of  Lowell  and  Lawrence  in  1890-91  seems  strongly 
to  demonstrate  that  even  a  small  number  of  virulent 
organisms  can  bring  about  an  almost  wholesale  infection. 
Indeed  if  the  virulent  organism  were  as  abundant  as  some 
recent  results  would  indicate  (Remlinger  and  Schneider, 
1897),  the  human  race  would  long  since  have  been  exter- 
minated. On  the  whole  it  seems  that  since  a  positive 
result  is  always  open  to  serious  doubt,  and  a  negative 
result  signifies  nothing,  the  search  for  the  typhoid  bacillus 
itself,  however  desirable  theoretically,  cannot  be  regarded 
at  present  as  generally  profitable. 

The  isolation  of  the  cholera  bacillus  from  water  can 


ISOLATION  OF  SPECIFIC  PATHOGEN ES.          55 

probably  be  accomplished  with  somewhat  less  difficulty 
than  is  encountered  in  the  case  of  B.  typhi.  Schottelius 
(Schottclius,  1885)  was  the  first  to  point  out  the  necessity 
for  growing  this  organism  in  an  alkaline  medium,  and 
Loeffler  (Loeffler,  1893)  found  that  its  isolation  from 
water  could  be  successfully  accomplished  by  adding  10  c.c. 
of  alkaline  peptone  broth  to  200  c.c.  of  the  infected  water 
and  incubating  for  twenty-four  hours  at  37°,  when  the 
organism  could  be  found  at  the  surface  of  the  medium. 

Somewhat  earlier  than  this  Dunham  (Dunham,  1887) 
had  made  a  special  study  of  the  chemical  reactions  of  the 
cholera  bacillus  and  found  that  the  organism  would  grow 
abundantly  in  a  solution  containing  i  per  cent  peptone 
and  .5  per  cent  salt  (Dunham's  solution),  producing  the 
"  cholera-red  or  nitroso-indol  reaction."  This  medium 
was  brought  into  practical  use  by  Dunbar  (Dunbar,  1892), 
who  succeeded  in  isolating  the  organisms  from  the  water 
of  the  Elbe  in  1892,  during  the  cholera  epidemic  at  Ham- 
burg. 

Koch  (Koch,  1893)  prescribed  the  following  method 
for  the  isolation  of  the  organism  from  water: 

To  ico  c.c.  of  the  water  to  be  examined  is  added  i 
per  cent  peptone  and  i  per  cent  salt.  The  mixture  is 
then  incubated  at  37°.  After  intervals  of  ten,  fifteen,  and 
twenty  hours  the  solution  is  examined  microscopically 
for  comma-shaped  organisms,  and  agar  plate  cultures  are 
made  which  are  likewise  incubated  at  37°.  If  any 
colonies  showing  the  characteristic  appearance  of  the 
cholera  bacillus  are  found,  these  are  examined  micro- 


56  ELEMENTS  OF  WATER  BACTERIOLOGY. 

scopically,  and  if  comma-shaped  organisms  are  present, 
inoculations  are  made  into  fresh  tubes  to  be  further  tested 
by  means  of  the  indol  reaction  and  by  inoculation  into 
animals.  The  existence  of  other  spirilla  of  some  patho- 
genic power  renders  necessary  the  greatest  care  and 
caution  in  claiming  positive  isolations.  That  no  great 
improvement  on  Koch's  method  has  been  made  during 
the  last  ten  years  seems  apparent  from  the  statements  of 
Kolle  and  Gotschlich  (Kolle  and  Gotschlich,  1903),  who 
employed  "the  peptone  method  with  subsequent  agar 
cultivation"  in  the  isolation  of  the  organisms  from  faeces 
of  cholera  patients  during  the  epidemic  in  Egypt  in  1902, 
and  who  have  made  notable  epidemiological  and  clinical 
researches  upon  this  disease. 

Other  pathogenic  organisms  have  been  isolated  from 
waters,  according  to  the  accounts  of  numerous  investi- 
gators, but  from  the  sanitary  point  of  view  the  typhoid 
and  cholera  bacilli  are  of  most  importance  since  these  are 
manifestly  the  germs  of  disease  most  likely  to  be  dissemi- 
nated through  this  medium.  For  the  detection  of  B. 
anthracis  and  other  spore-forming  pathogenic  bacteria 
which  may  at  times  gain  access  to  water  from  stockyards, 
slaughter-houses,  etc.,  the  method  suggested  by  Frank- 
land  (Frankland,  1894)  may  be  adopted.  The  water 
to  be  examined  is  heated  to  90°  for  two  minutes  and  then 
plated,  the  characteristic  colonies  of  the  anthrax  organism 
being  much  more  easily  discerned  after  the  destruction  of 
the  numerous  non-sporing  water  bacteria.  Again,  water 
is  sometimes  the  means  of  distributing  the  germs  of 


ISOLATION   OF  SPECIFIC  PATHOGEN ES.  57 

dysentery  and  diarrhoea,  as  shown  by  the  decrease  of 
these  diseases  in  Burlington,  Vt.  (Sedgwick,  1902),  and 
other  communities  where  pure  water-supplies  have  been 
substituted  for  polluted  ones.  It  is  possible  that  the 
examination  of  water  for  the  B.  dysenteriae  may  in  the 
future  help  to  throw  important  light  on  the  sanitary  con- 
dition of  a  water. 


CHAPTER  VI. 

METHODS  FOR  THE  ISOLATION  OF  THE 
COLON  BACILLUS. 

THE  Bacillus  coli  was  first  isolated  by  Escherich 
(Escherich,  1884)  from  the  faeces  of  a  cholera  patient. 
It  was  subsequently  found  to  be  a  normal  inhabitant  of 
the  intestinal  tract  of  man  and  many  other  animals  and 
to  occur  regularly  in  their  excretions,  and  on  this  account 
it  became  of  the  highest  interest  and  importance  to 
sanitarians,  since  its  presence  in  water-supplies  was 
regarded  as  direct  evidence  of  sewage  pollution. 

This  organism  may  be  described  as  a  short,  usually 
motile  rod,  with  diameter  generally  less  than  one  micron 
and  exhibiting  no  spore  formation.  It  forms  thin  irregu- 
lar translucent  films  upon  the  surface  of  gelatin,  called 
"grape-leaf  colonies"  by  the  Germans,  produces  no 
liquefaction,  and  gives  a  wire-nail-like  growth  in  stick 
cultures.  It  forms  a  white  translucent  layer  of  character- 
istic appearance  upon  agar,  produces  a  more  or  less 
abundant,  moist,  yellowish  growth  on  potato,  and  tur- 
bidity and  some  sediment  in  broth;  it  ferments  dextrose 
and  lactose  with  the  formation  of  gas  of  which  the  ratio  is 

H      2 

p^-  =— ,  according  to  most  investigators;    one  variety 

V'V-'g         I 

58 


ISOLATION  OF   THE  COLON  BACILLUS.  59 

TT  _ 

ferments  saccharose  with  a  gas  ratio  ^-  approaching-, 


and  another  does  not.  As  a  rule,  a  strong  acid  reaction 
is  developed  in  all  sugar-containing  media.  The  or- 
ganism reduces  nitrates  to  nitrites  and  sometimes  to 
ammonia.  It  coagulates  casein  in  litmus  milk,  and 
reduces  the  litmus  with  subsequent  slow  return  of  the 
color  (red),  and  forms  indol  in  peptone  solution.  Many 
cultures  of  this  organism  are  fatal  to  guinea-pigs  when 
the  latter  are  inoculated  subcutaneously  with  I  c.c.  of  a 
twenty-four-hour  bouillon  culture,  and  most  cultures 
produce  death  when  this  amount  is  inoculated  intraperi- 
toneally.  Although  not  a  spore-forming  bacillus,  and  in 
general  not  possessing  great  resistance  against  anti- 
septic substances,  B.  coli  seems  to  be  less  susceptible  to 
phenol  than  are  many  other  forms,  especially  certain 
water-  bacteria. 

The  Wurtz  litmus-lactose-agar  plate  (Wurtz,  1892),  as 
noted  in  Chapter  IV,  furnishes  one  ready  method  for  the 
isolation  of  B.  coli  from  water,  and  it  was  used  by  Sedg- 
wick  and  Mathews  for  the  purpose  as  early  as  1893 
(Mathews,  1893).  The  process  is  based  upon  the  fact 
already  alluded  to,  that  B.  coli  readily  ferments  lactose 
with  the  formation  of  acid.  If,  therefore,  plates  are  made 
with  agar  containing  both  lactose  and  litmus,  the  colon 
colonies  develop  as  red  spots  in  a  blue  field.  Since  or- 
ganisms other  than  B.  coli  may  also  develop  red  colonies, 
it  becomes  necessary  further  to  examine  the  red  colonies 
in  order  to  prove  that  they  are  made  up  of  colon  bacilli. 


60  ELEMENTS  OF   WATER  BACTERIOLOGY. 

This  is  done  by  fishing  from  isolated  suspicious-looking 
colonies,  replating  and  inoculating  into  the  usual  media 
for  diagnostic  work. 

For  success  in  examining  polluted  waters  by  this  method 
it  is  necessary  to  get  a  sufficient  dilution  so  that  colonies 
may  be  well  isolated,  and  to  this  end  it  is  advisable  that  a 
number  of  different  dilutions  be  employed,  a  series  of 
plates  being  prepared  from  each.  Under  any  conditions 
the  detection  of  the  colon  bacillus  is  seriously  hampered 
by  the  development  of  other  forms.  Certain  observers 
have  therefore  added  phenol  to  the  agar  medium,  com- 
bining the  effect  of  high  temperature  and  an  antiseptic 
to  check  the  growth  of  water-bacteria.  Copeland  for 
this  purpose  added  to  his  tubes  .2  c.c.  of  a  2%  solution 
of  phenol  (Copeland,  1901).  Chick  (Chick,  1900) 
found  that  1.33  parts  of  phenol  in  1000  materially  de- 
creased the  number  of  colon  bacilli  which  would  develop, 
while  i  part  gave  very  satisfactory  results,  the  plates 
showing  pure  cultures  of  B.  cohV 

The  test  for  the  colon  bacillus  may,  however,  be  made 
still  more  delicate  by  a  preliminary  enrichment  of  the 
sample  by  growth  in  a  liquid  medium  for  twenty-four 
hours  at  37°,  thus  greatly  increasing  the  proportion  of 
these  organisms  present  before  plating.  As  suggested 
in  the  classic  researches  of  Theobald  Smith  (Smith,  1892), 
this  method  may  be  made  approximately  quantitative  by 
the  inoculation  of  a  series  of  tubes  with  measured  por- 
tions of  the  water.  If,  for  example,  of  ten  tubes  inocu- 
lated each  with  ^  of  a  cubic  centimeter,  four  show 


x*u*  * 

/  Or 


¥N<VEK5ITY 

ISOLATION  OF.  THR  COLON  BACILLUS.  61 

the  B.  coli,  we  may  assume  that  some  40  of  these  organ- 
isms were  present  to  the  cubic  centimeter.  Irons  (Irons, 
1901),  in  a  comparative  study  of  various  methods  for  the 
isolation  of  B.  coli,  showed  that  the  preliminary  enrich- 
ment frequently  gave  positive  results  when  the  results 
of  the  direct  use  of  the  agar  plate  were  negative  and 
concluded  that  "  where  the  waters  to  be  examined  are 
much  polluted,  or  where  the  amount  of  B.  coli  is  small 
and  the  colony  count  large,  the  lactose  plate  for  plating 
water  direct  is  inferior  to  the  dextrose  fermentation- 
tube."  Gage  obtained  similar  results  (Gage,  1902). 
The  method  of  direct  plating  in  phenol-agar  is  quicker 
and  there  is  a  certain  danger  that  colon  bacilli  originally  _ 
present  may  be  overgrown  and  killed  out  in  the  enrich- 
ment process.  If  the  incubation  is  not  too  prolonged, 
however,  this  need  not  occur. 

The  medium  ordinarily  used  for  the  preliminary  enrich- 
ment is  ordinary  broth  to  which  2.5  per  cent  of  dextrose 
has  been  added,  and  the  reaction  brought  to  the  neutral 
point.  Into  each  of  a  number  of  fermentation-tubes  of  this 
medium  a  measured  quantity  of  the  water  to  be  examined 
is  inoculated,  and  the  culture  is  incubated  for  twenty-four 
hours  at  37.5°  C.  At  the  end  of  this  time  the  tubes  are 
examined  for  gas  formation.  If  gas  is  found,  a  small 
amount  of  the  culture  should  be  added,  after  suitable 
dilution,  to  litmus  lactose  agar  and  plated. 

With  polluted  waters  it  will  be  found  advantageous  to 
plate  out  on  the  first  appearance  of  gas  (4-8  hours).  It 
has  been  shown  by  one  of  us  (Prescott,  i902b)  that  a  very 


62  ELEMENTS  OF   WATER  BACTERIOLOGY. 

rapid  development  of  B.  coli  takes  place  in  the  first  few 
hours  after  dextrose  solutions  are  inoculated  with  intes- 
tinal material,  and  a  nearly  pure  growth  of  colon  bacilli 
often  results,  while  other  bacteria  multiply  more  slowly. 
With  highly  polluted  waters  gas  formation  will  probably 
begin  within  twelve  hours,  but  with  fewer  colon  bacilli 
present  the  duration  must  be  increased.  If  the  period 
of  incubation  be  too  long  continued,  trouble  in  the  subse- 
quent steps  of  the  isolation  may  be  encountered  because 
of  the  overgrowth  of  B.  coli  by  the  sewage  streptococci, 
which  are  almost  invariably  present,  and  which  by  their 
greater  acid-producing  powers  may  check  the  growth  of 
the  colon  bacilli.  . 

As  has  already  been  stated,  phenol  has  less  inhibitory 
action  upon  B.  coli  than  upon  normal  water-bacteria, 
hence  a  broth  containing  this  substance  may  be  employed 
for  preliminary  enrichment;  and  this  medium  has  been 
used  in  place  of  dextrose  broth  for  many  of  the  studies 
made  in  connection  with  the  Chicago  drainage  canal 
(Reynolds,  1902).  Phenol  broth  consists  of  ordinary 
broth  to  which  o.i  per  cent  phenol  is  added,  and  the 
method  of  procedure  is  to  add  i  c.c.  of  the  water  to  10  c.c. 
of  the  sterilized  phenol  broth  and  incubate  at  body 
temperature  for  twenty-four  hours.  Litmus-lactose-agar 
plates  are  then  made  and  the  examination  of  the  red 
colonies  carried  on  as  described  for  the  dextrose-broth 
method.  The  dextrose  broth  furnishes,  however,  a  much 
more  delicate  test  than  the  carbol  broth  when  the  num- 


ISOLATION  OF   THE  COLON  BACILLUS. 


ber  of  colon  bacilli  present  is  small,  as  is  clearly  shown  by 
the  following  table  from  Irons. 

PROPORTION  OF  POSITIVE  RESULTS  IN  TESTS  OF  POLLUTED  AND 
UNPOLLUTED  WATERS  BY  DEXTROSE  FERMENTATION-TUBE  AND 
CARBOL-BROTH  METHODS. 

(Irons,  1901.) 


Dextrose 
Fermentation- 
tube. 

Carbol-broth 
Method. 

Polluted  waters  •  

+       -         ? 
^•7      31        e 

+       -        ? 
?8     30      i 

Relatively  unpolluted  waters  .  .  . 

r6       ?8       2Z 

•77       6l        21 

Furthermore,  the  dextrose  fermentation-tube  possesses 
a  second  advantage  in  the  fact  that  a  decisive  negative 
test  may  be  obtained  within  twenty-four  hours  of  the 
original  planting  of  the  sample.  If  no  gas  is  formed  in 
the  tube,  we  commonly  assume  that  B.  coli  is  absent  and 
carry  the  examination  no  farther. 

When  it  is  desired  to  examine  samples  larger  than  i  c.c. 
for  B.  coli  it  becomes  necessary  to  modify  the  enrichment 
process  by  adding  the  nutrient  material  to  the  water 
instead  of  the  reverse.  For  this  purpose  phenol  dex- 
trose broth  (consisting  of  broth  with  10  per  cent  dex- 
trose, 5  per  cent  peptone,  and  .25  per  cent  phenol)  may 
be  added  to  the  sample  of  water  to  be  enriched  as  sug- 
gested by  Gage  (Gage,  1901).  Generally  10  c.c.  of  the 
broth  is  added  to  100  c.c.  of  the  water.  The  sample  is 
then  incubated  at  37°  for  twenty-four  hours,  and  if  at  the 
end  of  that  time  growth  has  taken  place,  a  cubic  centi- 


64  ELEMENTS  OF  WATER  BACTERIOLOGY. 

meter  is  inoculated  inte  a  dextrose  tube.  If  this  tube 
shows  gas  formation  after  twenty-four  hours  at  37°,  a  lit- 
mus-lactose-agar  plate  is  made  and  the  other  diagnostic 
tests  applied. 

Our  experience  has  shown  that  it  is  not  specially  advan- 
tageous to  apply  the  colon  test  in  such  large  samples, 
since  the  significance  of  B.  coli  when  present  in  numbers 
less  than  i  per  c.c.  is  extremely  doubtful.  On  the  other 
hand  the  danger  of  overgrowth  is  greatly  increased  by 
this  process  and  negative  results  may  often  be  obtained 
when  the  organisms  are  really  present.  Hunnewell  and 
one  of  ourselves  (Winslow  and  Plunnewell,  i902b)  found 
that  of  48  samples  of  certain  polluted  river  waters  18  gave 
B.  coli  when  i  c.c.  was  inoculated  directly  into  dextrose 
broth,  while  in  only  4  cases  was  a  positive  result  obtained 
after  preliminary  treatment  of  100  c.c.  in  carbol  broth. 
In  153  samples  from  presumably  unpolluted  water  B.  coli 
was  found  5  times  in  i  c.c.  and  1 1  times  by  the  examination 
of  the  larger  sample.  The  authors,  therefore,  concluded 
as  follows: 

"It  appears  evident  that  the  use  of  large  samples  in 
applying  the  colon  test  to  the  sanitary  analysis  of  drink- 
ing-water is  not  advantageous.  In  comparing  the  results 
of  the  tests  in  i  c.c.  and  in  ipo  c.c.,  it  will  be  noted  that  the 
proportion  of  lactose  fermenting  organisms  and  of  colon 
bacilli  in  the  unpolluted  waters  was  more  than  doubled 
in  the  latter;  thus  waters  of  good  quality  are  more  likely 
to  be  condemned  by  the  use  of  large  samples.  On  the 
other  hand,  in  the  polluted  waters  a  considerable  propor- 


ISOLATION  OF  THE  COLON  BACILLUS.  65 

tion  of  the  colon  bacilli  originally  present  were  lost  during 
the  incubation  of  the  large  samples,  so  that  waters  of  bad 
quality  actually  appeared  to  better  advantage  by  the  use 
of  100  c.c.  with  preliminary  incubation  in  phenol  broth." 

Similarly  Whipple  (Whipple,  1903)  notes  that  2.9  per 
cent  of  some  samples  of  water  examined  by  him  gave 
positive  tests  with  .1  c.c.  but  not  with  i  c.c.,  while  4.3  per 
cent  gave  positive  tests  with  .1  c.c.  or  i  c.c.  and  negative 
tests  with  10  c.c.  Again,  in  another  series  of  samples 
examined,  of  those  which  gave  positive  samples  in  smaller 
portions  5.3  per  cent  were  negative  in  10  c,c.,  4.7  per  cent 
in  loo  c.c.,  and  7.7  per  cent  in  500  c.c. 

In  all  practical  processes  of  examining  water  for  B.  coli 
one  essential  step  is  the  isolation  of  pure  cultures  upon 
the  litmus-lactose-agar  plate,  whether  the  plate  be  inocu- 
lated from  the  water  direct  or  from  a  preliminary  enrich- 
ment culture.  In  the  first  case  a  measured  quantity  of 
water  must  be  added.  In  the  second  case,  since  the 
enrichment- tube  was  inoculated  with  a  known  amount  of 
water  all  further  work  is  purely  qualitative  and  it  is  only 
necessary  to  obtain  such  a  number  of  colonies  upon  the 
lactose  plate  that  the  isolation  of  a  pure  culture  shall  be 
easy.  In  practice  the  following  procedure  has  been 
found  generally  successful.  After  the  dextrose-tubes  have 
been  incubated  for  twelve  to  twenty-four  hours  at  37°, 
from  those  which  show  gas,  one  loopful  is  carried  over 
to  a  tube  containing  10  c.c.  of  sterile  water,  and  of  this 
water  one  loopful  is  taken  for  the  inoculation  of  the  plate. 
Ordinarily  this  will  give  colonies  which  are  sufficiently 


66  ELEMENTS  OF  WATER  BACTERIOLOGY. 

well  separated,  but  a  second  plate,  inoculated  from  the 
dilution  water  with  a  straight  needle  instead  of  a  loop, 
furnishes  a  desirable  safeguard. 

The  litmus-lactose-agar  plates  made  in  this  manner 
should  be  incubated  for  from  twelve  to  twenty-four  hours  at 
the  body  temperature  (37°),  at  the  end  of  which  time  if  B. 
coli  is  present  red  colonies  upon  a  blue  field  will  be  visible. 
If  a  pure  culture  of  B.  coli  is  obtained,  the  litmus-lactose- 
agar  plate  may  become  blue  again  after  forty-eight  hours 
owing  to  the  formation  of  amines  and  ammonia  by  the 
action  of  the  bacteria  upon  the  nitrogenous  matter  present. 
If  the  dilution  is  too  low,  the  resulting  colonies  will  be 
small  and  imperfectly  developed,  making  it  difficult  to  be 
sure  of  pure  cultures  for  the  subsequent  tests.  A  great 
number  of  colonies  will  also  prevent  the  change  of  reac- 
tion from  acid  back  to  alkaline.  Since  many  bacteria 
ferment  lactose  with  the  formation  of  acid,  it  is  erroneous 
to  regard  all  colonies  as  those  of  B.  coli;  in  all  cases 
several  colonies  from  each  plate  should  be  isolated  upon 
agar  streaks  and  further  studied  in  subculture. 

In  the  selection  of  those  red  colonies  which  are  to  be 
fished  from  the  litmus-lactose-agar  plate  the  appearance 
of  the  growths  must  be  closely  noted.  A  colony  of  irregu- 
lar contour,  surrounded  by  a  very  faint  area  of  reddening, 
will  probably  belong  to  some  member  of  the  B..  mycoides 
group  (Winslow  and  Nibecker,  1903);  small,  compact, 
bright-red  colonies  are  characteristic  of  the  streptococci, 
and  Gage  and  Phelps  (Gage  and  Phelps,  1903)  have 
pointed  out  that  of  these  there  are  two  types,  one  of  a 


ISOLATION   OF   THE  COLON  BACILLUS.  67 

brick- red  color,  and  of  such  consistency  as  to  be  readily 
picked  up  by  the  needle-point  and  the  other  smaller  and 
of  an  intense  vermilion  color.  The  colonies  of  the  colon 
bacillus  are  usually  well  formed,  pulvinate  on  the  sur- 
face and  fusiform  when  growing  deeper  down. 

The  agar  streak  made  from  the  litmus-lactose-agar  plate 
shows  after  twenty-four  hours  certain  marked  character- 
istics. The  most  distinct  types  are  two,  the  abundant, 
first  translucent,  later  whitish  and  cheesy  growth,  cover- 
ing nearly  the  whole  surface  of  the  agar,  characteristic  of 
B.  coli  and  its  allies,  and  a  very  faint  growth,  either  con- 
fined strictly  to  the  streak  or  made  up  of  faint  isolated 
colonies,  dotted  here  and  there  over  the  surface.  The 
latter  cultures  are  typical  of  the  sewage  streptococci,  and 
a  microscopic  examination  will  generally  settle  their  status 
at  once.  Of  the  more  luxuriant  growths,  some  of  which 
are  stringy  to  the  needle  many  will  generally  prove  to  be 
atypical,  and  if  any  of  the  weakly  fermenting  forms  (B. 
mycoides)  are  present,  a  dull  wrinkled  growth  will  be 
produced. 

Having  submitted  the  sample  of  suspected  water  to  a 
preliminary  enrichment  process,  and  having  isolated  pure 
cultures  of  suspicious  organisms  from  the  litmus-lactose- 
agar  plate,  the  third  step  is  the  examination  of  the  specific 
reactions  of  the  organisms  thus  obtained.  Just  what 
characters  to  use  in  defining  the  " colon  bacillus"  is  a 
matter  of  prime  importance.  The  whole  question  of 
species  among  the  bacteria  is  an  extremely  complex  one, 
since  around  each  definite  species  are  grouped  forms 


68  ELEMENTS  OF   WATER  BACTERIOLOGY. 

differing  from  the  type  in  one  or  two  of  its  character- 
istics. 
As  Whipple  says   (Whipple,  1903),  "The  type  form  of 

Bacillus  coli  is  one  which  can  be  defined  within  reason- 
ably narrow  limits,  but  when  the  organism  has  been  away 
from  its  natural  habitat  for  varying  periods  of  time,  and 
has  existed  under  abnormal  conditions,  its  ability  to  react 
normally  to  the  usual  tests  appears  to  be  greatly  impaired. 
Its  power  to  reduce  nitrates  may  be  lost,  or  on  the  other 
hand  may  be  increased;  its  power  to  produce  indol  may 
be  lost,  or  on  the  other  hand,  it  may  be  increased;  its 
power  to  coagulate  milk,  even,  is  sometimes  reduced, 
although  seldom  entirely  lost;  its  power  to  ferment  car- 
bohydrates may  be  altered  so  that  the  amount  of  gas 
obtained  in  a  fermentation-tube,  as  well  as  its  ratio  of 
H  to  CO2,  is  quite  abnormal.  But  in  spite  of  all  these 
facts,  the  bacillus  tested  may  have  been  originally  a  true 
Bacillus  coli." 

The  more  of  such  atypical  forms  which  are  included 
the  greater  will  be  the  number  of  positive  isolations.  At 
present  our  definitions  must  be  more  or  less  arbitrary; 
each  observer  will  consider  as  true  colon  bacilli  those 
which  fulfil  his  particular  set  of  tests,  and  will  class  as 
pseudo-colon  organisms  those  which  do  not.  If  we  find, 
having  established  such  an  arbitrary  standard,  that  the 
colon  bacillus,  as  determined  by  it,  is  found  in  waters 
known  to  be  polluted,  and  not,  as  a  rule,  in  those  known 
to  be  free  from  pollution,  the  sanitarian  can  afford  to 
ignore  the  theoretical  question  of  specific  values  and 


ISOLATION  OF   THE  COLON   BACILLUS.  69 

make  confident  use  of  the  practical  test.  It  is,  of  course, 
highly  desirable  that  some  standard  set  of  reactions 
should  be  commonly  adopted  by  sanitary  bacteriologists; 
and  it  is  to  be  hoped  that  some  such  uniform  scheme  may 
soon  be  drawn  up  by  the  Society  of  American  Bacteriolo- 
gists or  by  the  American  Public  Health  Association.  At 
present  the  plan  developed  by  the  Massachusetts  State 
Board  of  Health  (Massachusetts  State  Board  of  Health, 
1899)  is  most  widely  prevalent  in  this  country.  It  in- 
volves the  use  of  six  simple,  definite,  positive  tests — the 
growth  in  gelatin,  lactose-agar,  dextrose  broth,  milk, 
nitrate  solution,  and  peptone  solution. 

For  recording  the  results  of  the  various  tests  applied, 
the  appended  blank  form  has  been  in  use  at  the  Massa- 
chusetts Institute  of  Technology.  On  the  upper  part  of 
the  sheet  are  noted  the  results  of  the  gelatin  count  and 
the  litmus-agar  count  at  37°.  In  the  second  case  the 
number  of  acid-formers  is  placed  in  brackets  after  the 
total  numbers.  The  lower  Dart  is  used  for  the  B.  coli 
isolation. 

MASSACHUSETTS  INSTITUTE  OF  TECHNOLOGY. 

BACTERIOLOGICAL  EXAMINATION  OF  WATER. 
Sample  No.  Date  of  collection 

Examined  by  Hour  of  collection 

Place  of  collection  Remarks 

QUANTITATIVE  ANALYSIS. 

Gelatin-plate  cultures.  No.  colonies       Agar-plate  cultures   at   37- 

48  hours  per  c.c.  No.  colonies  per  c.c. 

24  hours 

Average  Average 

Remarks  Remarks 


70  ELEMENTS  OF   WATER  BACTERIOLOGY. 

QUALITATIVE   ANALYSIS. 

Preliminary  fermentation -tube 

Litmus-lactose-agar  plates 

Agar  streak  culture 

Fermentation-tube 

Nitrate  test 

Milk  test 

Indol  reaction 

Gelatin  stab  culture 

Remarks 

Opposite  the  words  "  Preliminary  f ermentation-tube  " 
the  results  of  the  first  enrichment  test  in  the  five  or  ten 
tubes  inoculated  are  recorded  by  plus  or  minus  signs. 
Under  the  columns  headed  by  plus  signs  the  presence  or 
absence  of  typical  red  colonies  on  the  litmus-lactose-agar 
plate  is  similarly  indicated.  The  third  line  serves  for 
the  results  of  the  microscopic  and  macroscopic  appear- 
ance of  the  agar  streak.  If  this  gives  the  proper  type  of 
growth  and  rod-shaped  organisms  are  found,  a  dex- 
trose fermentation-tube,  a  nitrate  tube,  a  milk  tube,  a 
peptone  tube,  and  a  gelatin  stab  are  inoculated  from  it. 

After  twenty- four  hours  incubation  at  37°,  the  first 
three  of  these  subcultures  are  examined  and  the  results 
recorded  in  the  proper  columns  by  plus  and  minus  signs. 
The  amount  of  gas  in  the  closed  arm  of  the  dextrose  tube 
may  be  conveniently  measured  by  the  Frost  gasometer 
(Frost,  1901).  Then  a  few  centimeters  of  strong  sodium 
or  potassium  hydrate  are  added  and  mixed  with  the  broth 
by  cautiously  tipping  the  tube  and  a  second  measurement 
determines  the  amount  of  gas  absorbed  (assumed  to  be 
CO2).  The  gas  should  first  fill  from  a  third  to  two- 


ISOLATION  OF   THE  COLON  BACILLUS.  71 

thirds  of  the  closed  arm  and  about  one-third  of  this  should 
be  absorbed.  The  nitrate  tube  is  tested  for  nitrites  by 
adding  a  drop  of  the  following  solutions  in  succession: 

A.  Sulphanilic  acid 5  gram 

Acetic  acid  (25%  sol.) 150.0  c.c. 

B.  Naphthylamine  chloride .1  gram 

Distilled  water 20.0  c.c. 

Acetic  acid  (25%  sol.) 150.0  c.c. 

A  red  or  violet  coloration  indicates  the  presence  of 
nitrites.  The  milk  tube  is  merely  heated  to  boiling  over 
the  free  flame ;  if  coagulation  occurs  the  test  is  considered 
positive. 

After  seventy- two  hours  incubation  at  37°  the  peptone 
solution  is  examined  for  indol  by  adding  i  c.c.  of  a  .02  per 
cent  solution  of  sodium  or  potassium  nitrite  and  i  c.c.  of  a 
i  to  i  solution  of  sulphuric  acid.  Both  the  tube  and 
the  reagents  should  be  cooled  on  ice  before  mixing,  and 
the  tube  should  be  left  in  a  cool  place  for  an  hour  after- 
ward to  allow  time  for  the  characteristic  rose- red  color 
of  nitroso-indol  to  develop.  The  gelatin  tube  is  kept  at 
20°  for  seven  days  according  to  the  procedure  adopted 
at  the  Massachusetts  Institute  of  Technology.  It  seems 
undesirable  in  practice  to  prolong  the  test  much  beyond 
this  point,  although  some  slowly  liquefying  organisms 
are  doubtless  included,  which  would  be  thrown  out  by  a 
longer  incubation.  The  extent  of  this  source  of  error 
as  well  as  the  relative  importance  of  the  various  other 
diagnostic  tests  is  well  shown  in  the  following  table  of 


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ELEMENTS   OF   WATER   BACTERIOLOGY. 


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ISOLATION  OF   THE  COLON  BACILLUS.  73 

the  results  obtained  at  Lawrence  during  a  period   of 
eighteen  months. 

It  should  be  noted  that  considerable  differences  often 
appear  in  these  biochemical  reactions  between  tubes  of 
the  same  batch  of  a  medium,  inoculated  with  approxi- 
mately the  same  amount  of  the  same  culture.  This  has 
been  shown  very  markedly  for  the  amount  of  the  gas  and 
the  proportion  of  carbon  dioxide  in  the  dextrose  tube  by 
Fuller  and  Johnson  (Fuller  and  Johnson,  1899),  Penning- 
ton  and  Kiisel  (Pennington  and  Kiisel,  1900),  Gage 
(Gage,  1902),  and  one  of  ourselves  (Winslow,  1903). 
Variations  in  the  nitrate  reduction  are  often  even  more 
marked,  one  tube,  perhaps,  showing  a  strong  reaction 
and  another  none.  In  important  cases,  therefore,  it  is 
desirable  to  inoculate  the  subcultures  in  duplicate. 


CHAPTER  VII. 
SIGNIFICANCE  OF  THE  PRESENCE  OF  B.  COLI  IN  WATER. 

TEN  years  ago  the  B.  coli  of  Escherich  occupied  a  posi- 
tion of  very  great  prominence  in  the  eyes  of  sanitarians. 
If  it  was  not  considered  to  be  in  itself  a  dangerously  patho- 
genic germ,  it  was  at  least  regarded  as  a  suspiciously  close 
relation  of  the  typhoid  organism.  At  this  time,  therefore, 
the  alleged  presence  of  either  of  these  forms  was  quite 
sufficient  to  condemn  a  water-supply. 

Investigation  soon  showed,  however,  that  the  Bacillus 
coli  was  by  no  means  confined  to  the  human  intestine. 
Dyar  and  Keith  (Dyar  and  Keith,  1893)  found  it  to  be  the 
prevailing  intestinal  form  in  the  cat,  dog,  hog,  and  cow. 
In  the  goat  and  rabbit  they  reported  that  no  single  organ- 
ism was  constantly  present;  and  in  the  case  of  the  horse, 
the  place  of  the  colon  bacillus  was  taken  by  a  new  form, 
described  by  the  authors  under  the  name  of  B.  equi  intes- 
tinalis.  About  the  same  time,  Fremlin  (Fremlin,  1893) 
found  colon  bacilli  in  the  feces  of  dogs,  mice,  and  rabbits, 
but  not  in  those  of  rats,  guinea-pigs,  and  pigeons.  Smith 
(Smith,  1895)  recorded  the  presence  of  the  same  or- 
ganism, in  almost  pure  cultures,  in  the  intestines  of  dogs, 

74 


THE  SIGNIFICANCE  OF  B.  COLI  IN  WATER.      7$ 

cats,  swine,  and  cattle;  and  he  also  found  it  in  the  organs 
of  fowls  and  turkeys  after  death.  Brotzu  (Brotzu,  1895) 
reported  B.  coli  and  allied  forms  as  very  abundant  in  the 
intestine  of  the  dog;  and  Belitzer  (Belitzer,  1899)  still 
more  recently  isolated  typical  colon  bacilli  from  the 
intestinal  contents  of  horses,  cattle,  swine,  and  goats. 
Russell  and  Bassett  (Russell  and  Bassett,  1899)  stated  that 
studies  made  by  E.  B.  Hoag  in  Professor  Russell's  labora- 
tory indicated  the  presence  of  bacilli  of  the  colon  group, 
fermenting  dextrose  with  a  gas  formula  of  f ,  in  the  feces 
of  "  a  considerable  number  of  different  species  of  mam- 
malia, as  well  as  that  of  birds,  fish,  etc.,"  while  similar 
organisms  with  the  inverted  gas  formula  \  were  con- 
sidered by  the  same  authors  to  be  characteristic  of  decom- 
posing organic  matter  of  vegetable  origin,  free  from  sus- 
picion of  fecal  contamination.  Moore  and  Wright 
(Moore  and  Wright,  1900)  recorded  the  finding  of  the 
colon  bacillus  in  the  horse,  dog,  cow,  sheep,  and  hen; 
and  in  a  later  report  (Moore  and  Wright,  1902)  they  noted 
its  occurrence  in  swine  and  in  some,  but  not  all,  the  speci- 
mens of  rabbits  examined.  In  frogs  it  was  not  found. 
Amyot  (Amyot,  1902)  failed  to  find  B.  coli  in  the  intes- 
tines of  23  fish  representing  14  species.  The  possibility 
that  colon  bacilli  may  be  introduced  into  unpolluted 
waters  through  the  agency  of  fish  was  considered  again 
by  Johnson,  in  a  paper  read  at  the  Washington  meeting 
of  the  American  Public  Health  Association  and  not  yet 
published.  Colon  bacilli  were  found  present  in  the 
intestinal  tract  of  fish  taken  from  polluted  waters,  and 


76  ELEMENTS  OF   WATER   BACTERIOLOGY. 

the  conclusion  was  drawn  that  the  migration  of  fish  from 
a  contaminated  stream  or  lake  to  an  unpolluted  one  may 
explain  the  occasional  finding  of  B.  coli  in  small  samples, 
or  the  more  regular  detection  of  it  in  large  volumes  of  the 
water. 

Many  bacteriologists  have  gone  much  further  and 
affirmed  that  the  colon  bacillus  was  not  a  form  character- 
istic of  the  intestine  at  all,  but  a  saprophyte  having  a  wide 
distribution  in  nature.  The  first  of  this  school,  perhaps, 
was  Kruse  (Kruse,  1894), who  in  1894  protested  against  the 
arbitrary  conclusions  drawn  from  the  colon  test  as  then  ap- 
plied. He  pointed  out  that  the  characters  usually  observed 
marked,  not  a  single  species,  but  a  large  group  of  organisms. 
As  ordinarily  defined,  he  added,  "the  Bacterium  coli  is  in 
no  way  characteristic  of  the  feces  of  men  or  animals. 
Such  bacteria  occur  everywhere,  in  air,  in  earth,  and  in 
the  water,  from  the  most  different  sources."  Even  if  the 
relations  to  milk  and  sugar  media  be  considered,  "  micro- 
organisms with  these  characteristics  are  also  widespread." 
Dr.  Kruse  gave  no  experimental  data  on  which  his  opinion 
was  based.  In  the  same  year  Beckmann  (Beckmann, 
1894)  isolated  a  bacillus  which  he  identified  by  pretty 
thorough  tests  as  B.  coli  from  the  city  water  of  Strass- 
burg,  a  ground- water  which  he  believed  could  by  no  pos- 
sibility be  subject  to  fecal  contamination.  Large  quan- 
tities of  water  were  used  for  the  isolation. 

Refik  (Refik,  1896)  recorded  the  constant  presence  of 
colon  bacilli  in  water  of  all  sorts,  public  supplies,  wells, 
cisterns,  and  springs  in  the  neighborhood  of  Constant!- 


THE  SIGNIFICANCE  OF  B.  CO  LI  IN   WATER.       77 

nople,  but  the  only  characters  which  these  tl colon  bacilli" 
exhibited  in  common  were  the  "classical  growth"  upon 
potato,  the  possession  of  less  than  8  cilia,  and  the  power 
of  active  development  on  certain  media  upon  which  the 
typhoid  bacillus  did  not  grow.  A  more  careful  and  sig- 
nificant piece  of  work  on  the  same  line  was  published  by 
Poujol  in  the  succeeding  year.  This  author  reported 
(Poujol,  1897)  the  isolation  of  B.  coli  from  22  out  of  34 
waters  studied  by  him  in  relation  to  their  use  as  public 
supplies.  The  waters  were  from  various  sources — springs, 
wells,  and  rivers — but  all  were  of  fair  quality  and  many 
quite  free  from  any  possibility  of  contamination.  Samples 
of  100  c.c.  were  used  for  analysis;  in  the  only  case  in  which 
a  smaller  amount  was  also  tested,  broth  inoculated  with 
10  drops  of  the  water  and  placed  at  45°  C.  remained 
sterile.  The  author  concluded  that  "fecal  contamination 
can  only  exceptionally  be  invoked  to  explain  the  presence 
of  B.  coli  in  water.  As  the  bacteria  of  the  subterranean 
water  are  contributed  to  it  from  the  surface  of  the  earth 
by  the  water  which  filters  downward,  I  am  rather  inclined 
to  believe  in  a  general  diffusion  of  B.  coli  either  on  the 
surface  of  the  earth,  where  it  might  be  deposited  with 
the  dust  of  the  air,  or  in  the  superficial  layers  of  the  earth, 
which  may  form  one  of  its  normal  habitats."  Therefore, 
the  author  considered  that  caution  should  be  exercised 
in  condemning  a  water  on  account  of  the  presence  of  B. 
coli,  except,  as  he  added,  "for  those  cases  where  it  exists 
in  considerable  quantity." 

Certain  Italian  observers  appear  to  have  come  to  even 


7 8  ELEMENTS  OF   WATER  BACTERIOLOGY. 

less  conservative  conclusions.  Abba  (Abba,  1895)  found 
colon  bacilli  constantly  present  in  certain  unpolluted 
waters  near  Turin.  Moroni  (Moroni,  1898;  Moroni, 

1899)  reported  the  examination  of  numerous  deep  and 
shallow  wells  and  unpolluted  springs  about  Parma,  as 
well  as  of  the  public  water-supply  of  the  city,  for  the  colon 
bacillus  and  concluded  that  that  organism  was  a  water  form 
and  had  no  sanitary  significance.     The  characters  used 
for  the  identification  of  the  species  in  this  case  were  fairly 
exhaustive,  but  both  Abba  and  Moroni  used  liter  samples 
for  analysis. 

Levy  and  Bruns  (Levy  and  Bruns,  1899)  gave  a  new 
turn  to  the  discussion  by  emphasizing  the  importance  of 
animal  inoculation,  already  suggested  by  Blachstein  (Blach- 
stein,  1893)  and  others.  They  claimed  that  the  existence  of 
numerous  para-colon  and  para-typhoid  organisms  in  air,  in 
dust,  and  in  unpolluted  water  made  it  impossible  to  decide 
by  ordinary  bacteriological  methods  whether  true  colon 
bacilli  were  present  in  water  or  not.  In  no  case,  how- 
ever, did  representatives  of  the  colon  group  isolated  by 
them  from  water  kill  a  guinea-pig,  even  when  i  or  2  c.c. 
were  injected  intraperitoneally;  and  the  authors,  there- 
fore, considered  pathogenicity  as  an  attribute  belonging 
only  to  the  true  B.  coli  of  the  intestine.  This  paper 
aroused  Professor  Kruse's  pupil,  Weissenfeld,  to  a  pub- 
lication, in  which  the  position  of  the  Bonn  school  was 
carried  to  an  extreme.  Weissenfeld  reported  (Weissenfeld, 

1900)  the  analysis  of  30  samples  of  water  supposedly  pure, 
and  of  26  samples  considered  to  be  contaminated.     In 


THE  SIGNIFICANCE  OF  B.  CO  LI  IN   WATER.       79 

each  case  a  single  centimeter  sample  was  first  incubated 
in  Parietti  broth,  and  if  no  growth  occurred,  larger  samples 
of  half  a  liter  or  a  liter  were  examined.  Colon  bacilli 
were  found  in  all  the  samples  examined;  and  the  patho- 
genicity  varied  independently  of  the  source  of  the  water.1 
The  author  concluded  that  "the  so-called  Bacterium 
coli  may  be  found  in  waters  from  any  source,  good  or  bad, 
if  only  a  sufficiently  large  quantity  of  the  water  be  taken 
for  analysis."  In  the  first  place  it  should  be  noted  that 
the  characters  used  by  this  investigator  for  defining  the 
"so-called  Bacterium  coli"  were  absolutely  inadequate. 
He  classed  under  that  head  all  bacilli  of  medium  size, 
which  formed  grapevine-leaf  colonies  on  gelatin  and 
gas  in  sugar  agar,  which  were  more  or  less  motile,  or 
rarely  non-motile,  and  which  were  decolorized  by  the 
Gram  method.  As  regards  coagulation  of  milk  and 
formation  of  indol,  "the  bacteria  isolated  differed."  In 
the  second  place  it  is  difficult  to  see  how  the  author  could 
possibly  have  believed  that  his  experiments  proved  the 
isolation  of  the  colon  bacillus  to  be  "useless  as  an  aid  in 
the  sanitary  examination  of  water,"  as  the  title  of  the 
paper  runs.  Even  his  own  work  furnishes  strong  evi- 
dence to  the  contrary.  In  24  of  the  26  samples  from  bad 
sources,  he  isolated  his  imperfectly  defined  colon  bacilli 
from  i  c.c.  of  the  water,  while  in  only  8  of  the  30  samples 
of  good  waters  could  he  find  such  organisms  in  that 
quantity. 
Of  a  series  of  47  cultures  of  actic-acid  bacteria 

1  Confirmed  by  Savage  (Savage,  1903,. 


So  ELEMENTS  OF  WATER  BACTERIOLOGY. 

recently  examined  by  one  of  ourselves  (Prescott,  1902  a; 
Prescott,  1903)  25  were  found  to  give  the  reactions  of  B. 
coli.  These  organisms  were  isolated  chiefly  from  cereals 
and  products  of  milling,  such  as  flour,  bran,  corn-meal, 
oats,  barley,  etc.,  while  others  were  in  technical  use  for 
producing  the  lactic  fermentation.  There  is  no  evidence 
that  any  of  these  organisms  were  of  intestinal  origin,  and 
yet  they  possess  all  the  characters  of  typical  colon  bacilli, 
even  to  the  pathogenic  action  when  inoculated  into 
guinea-pigs.  These  results  explain  the  results  of  Klein 
and  Houston  (Klein  and  Houston,  1900),  who  reported 
the  finding  of  typical  colon  bacilli  in  3  out  of  24  samples 
of  wheat  and  oats  obtained  from  a  wholesale  house;  rice, 
flour,  and  oat- meal  bought  at  two  different  retail  shops 
gave  B.  coli  on  all  three  cereals  in  i  case  and  on  none  in 
the  other.  In  Germany,  Papasotiriu  (Papasotiriu,  1901) 
was  meanwhile  carrying  on  almost  exactly  similar  inves- 
tigations to  Prescott' s,  with  identical  results. 

What  then  does  a  colon  test  prove?  Obviously  (i)  the 
presence  of  a  colon  bacillus  which  has  come  directly  from 
the  intestine  of  some  animal,  or  (2)  of  a  colon  bacillus 
which  has  come  indirectly  from  an  animal,  or  (3)  of  a 
saprophytic  "lactic- acid  bacillus"  which  gives  the  same 
biochemical  reactions.  This  immediately  raises  the 
interesting  questions,  Is  it  possible  that  the  lactic-acid 
bacilli  have  been  indirectly  derived  from  animal  intes- 
tines, having  "  escaped  from  cultivation/'  as  the  botanists 
say?  Or  is  the  converse  true,  namely,  that  all  colon 
bacilli  are  simply  lactic-acid  bacteria  which  have  found 


THE  SIGNIFICANCE  OF  B.  COLI  IN  WATER.       81 

in  the  warm  intestinal  canal  richly  supplied  with  food  a 
favorable  habitat? 

The  answer  to  these  questions  is  of  much  theoretical 
interest,  but  need  not  be  further  considered  here.  The 
practical  sanitary  conclusions  to  be  drawn  are  as  follows: 

1.  Bacteria  corresponding  in  every  way  to  B.  coli  are 
by  no  means  confined  to  animal  intestines,  but  are  widely 
distributed  elsewhere  in  nature. 

2.  The  finding  of  a  few  colon  bacilli  in  large  samples 
of  water,  or  its  occasional  discovery  in  small  samples, 
does  not  necessarily  have  any  special  significance. 

3.  The  detection  of  B.  coli  in  a  large  proportion  of 
small  samples  (i  c.c.  or  less)  examined  is  imperatively 
required  as  an  indication  of  recent  sewage  pollution. 

4.  The  number  of  colon  bacilli  in  water  rather  than  their 
presence  should  be  used  as  a  criterion  of  recent  sewage 
pollution. 

With  these  qualifications  the  value  of  the  colon  test 
was  never  more  firmly  established  than  it  is  to-day. 
Whether  or  not  originally  a  domesticated  form,  it  is  clear 
that  the  colon  bacillus  finds  in  the  intestine  of  the  higher 
vertebrates  an  environment  better  suited  to  its  growth 
and  multiplication  than  any  other  which  occurs  in  nature. 
It  is  almost  certain  that  the  only  way  in  which  large 
numbers  of  these  organisms  gain  access  to  natural  waters 
is  by  pollution  with  the  domestic,  industrial,  and  agri- 
cultural wastes  of  human  life.  If  pollution  has  been 
recent,  colon  bacilli  will  be  found  in  comparatively  small 
samples  of  the  waters.  If  pollution  has  been  remote  the 


82  ELEMENTS  OF   WATER  BACTERIOLOGY. 

number  of  colon  bacilli  will  be  small,  since  there  is  good 
evidence  that  the  majority  of  intestinal  bacteria  die  from 
exposure  in  cold  water.  If  derived  from  cereals  or  the 
intestines  of  wild  animals  the  number  will  be  insignificant 
except  in  the  vicinity  of  great  grain-fields  or  when  the 
water  receives  refuse  from  grist-mills,  tanneries,  dairies, 
or  lactic-acid  factories. 

The  first  recognition  of  the  necessity  for  a  quantitative 
estimation  of  colon  bacilli  in  water  we  owe  to  Dr.  Smith, 
who  in  1892  (Smith,  1893^  outlined  a  plan  for  such  a 
study  to  be  made  by  the  New  York  Board  of  Health  on  the 
Mohawk  and  Hudson  Rivers.  Burri  (Burri,  1895)  pointed 
out  that  the  use  of  so  large  a  sample  as  a  liter  for  exam- 
ination would  lead  to  the  condemnation  of  many  good 
waters.  Freudenreich  (Freudenreich,  1895)  at  the  same 
time  indicated  the  necessity  for  taking  into  account  the 
number  of  colon  bacilli  present.  He  recorded  the  isolation 
of  the  organism  from  unpolluted  wells,  when  as  large  a 
quantity  of  water  as  100  c.c.  was  used,  and  concluded  that 
it  was  entirely  absent  only  from  waters  of  great  purity 
and  present  in  large  numbers  only  in  cases  of  high  pollu- 
tion. This  author  also  quoted  Miquel  as  having  found 
colon  bacilli  in  almost  every  sample  of  drinking-water  if 
only  a  sufficient  portion  were  taken  for  analysis,  but  gave 
no  reference. 

The  practical  results  of  the  application  of  the  colon 
test  from  this  standpoint  have  always  proved  most  instruc- 
tive. As  originally  outlined  by  Dr.  Smith,  it  consisted  in 
the  inoculation  of  a  series  of  dextrose  tubes  with  small 


THE  SIGNIFICANCE  OF  B.  COLI  IN  WATER.       83 

portions  of  water,  tenths  or  hundredths  of  the  cubic  centi- 
meter. It  was  first  used  by  Brown  (Brown,  1893)  in  1892 
for  the  New  York  State  Board  of  Health,  and  its  results 
showed  from  22  to  92  fecal  bacteria  per  c.c.  in  the  water 
of  the  Hudson  River  at  the  Albany  intake,  and  from  3  to 
49  at  various  points  in  the  Mohawk  River  between  Amster- 
dam and  Schenectady.  In  some  previous  work  at  St. 
Louis,  the  colon  bacilli  in  the  Mississippi  River  were  found 
to  vary  from  3  to  7  per  c.c. 

Hammerl  (Hammerl,  1897)  used  the  presence  of  Bacillus 
coli  as  a  criterion  of  self-purification  in  the  river  Mur. 
He  considered,  in  spite  of  the  position  taken  by  Kruse, 
that  when  a  water  contained  large  numbers  of  colon  bacilli, 
as  well  as  an  excess  of  bacteria  in  general,  it  might  be 
considered  to  be  contaminated  by  human  or  animal  excre- 
ment. As,  however,  the  organism  would  naturally  be 
present  in  large  quantities  of  such  a  water  as  that  of  the 
Mur,  he  used  no  enrichment  process,  but  made  plate  cul- 
tures direct;  he  defined  the  B.  coli  as  a  small  bacillus, 
non-motile  or  but  feebly  motile,  growing  rapidly  at  37°  C., 
coagulating  milk  and  forming  gas  in  sugar  media.  In 
general,  Hammerl  failed  to  find  colon  bacilli  in  the 
river  by  this  method,  except  immediately  below  the 
various  towns  situated  upon  it ;  at  these  points  of  pollution 
he  discovered  a  few  colon  colonies  upon  his  plates,  not 
more  than  4  to  6  per  c.c.  of  the  water.  He  concluded 
that  "the  Bacterium  coli,  even  when  it  is  added  to  a 
stream  in  great  numbers,  under  certain  circumstances 
disappears  very  rapildy,  so  that  it  can  no  longer  be  detected 


84  ELEMENTS  OF  WATER  BACTERIOLOGY. 

in  the  examination  of  small  portions  of  the  water."  It 
should  be  noted  that  Hammerl' s  method  was  much  less 
delicate  than  the  use  of  the  dextrose  tube  for  preliminary 
incubation. 

A  very  important  series  of  observations  carried  out  in 
England  by  the  bacteriologists  of  the  local  government 
board  has  led  to  similar  conclusions.  Dr.  Houston  in 
particular  (Houston,  1898;  Houston,  i899a;  Houston, 
i9coa)  made  an  elaborate  series  of  examinations  of  soils 
from  various  sources  to  see  whether  the  microbes  con- 
sidered to  be  characteristic  of  sewage  could  gain  access 
to  water  from  surface  washings  free  from  human  con- 
tamination. In  the  three  papers  published  on  this  sub- 
ject the  examination  of  46  soils  was  recorded.  In  only 
10  of  the  samples  was  B.  coli  found,  and  of  these  10,  9 
were  obviously  polluted,  being  derived  from  sewage  fields, 
freshly  manured  land,  or  the  mud-banks  of  sewage-pol- 
luted rivers.  The  author  finally  concluded  that  "as  a 
matter  of  actual  observation,  the  relative  abundance  of 
B.  coli  in  pure  and  impure  substances  is  so  amazingly 
different  as  to  lead  us  to  suspect  that  not  only  does  B.  coli 
not  flourish  in  nature  under  ordinary  conditions,  but  that 
it  tends  to  even  lose  its  vitality  and  die."  "  In  brief,  I  am 
strongly  of  opinion  that  the  presence  of  B.  coli  in  any  num- 
ber, whether  in  soil  or  in  water,  implies  recent  pollution  of 
animal  sort.''  Pakes  (Pakes,  1900)  stated  on  the  strength 
of  an  examination  of  "  about  300  different  samples  of 
water,"  no  particulars  being  published,  that  water  from 
a  deep  well  should  not  contain  B.  coli  at  all,  but  that  water 


THE  SIGNIFICANCE  OF  B.  CO  LI  IN   WATER.       85 

from  other  sources  need  not  be  condemned  unless  the 
organism  was  found  in  20  c.c.  or  less.  When  colon  bacilli 
were  fotind  in  only  greater  quantities  than  100  c.c.  the 
water  might  be  considered  as  probably  safe.  Horrocks 
(Horrocks,  1901),  after  a  general  review  of  English  prac- 
tice, concluded  that  "when  a  water-supply  has  been 
recently  polluted  with  sewage,  even  in  a  dilution  of  one  in 
one  hundred  thousand,  it  is  quite  easy  to  isolate  the  B.  coli 
from  i  c.c.  of  the  water."  "I  would  say  that  a  water 
which  contained  B.  coli  so  sparingly  that  200  c.c.  required 
to  be  tested  in  order  to  find  it  had  probably  been  polluted 
with  sewage,  but  the  contamination  was  not  of  recent 
date."  Chick  (Chick,  1900)  found  6100  colon  bacilli 
per  c.c.  in  the  Manchester  ship  canal,  55-190  in  the  pol- 
luted River  Severn,  and  numbers  up  to  65,000  per  gram  in 
roadside  mud.  On  the  other  hand,  of  38  unpolluted 
streams  and  rivulets,  31  gave  no  Bacillus  coli  a*hd  the 
other  7  gave  i  per  c.c.  or  less.  The  Liverpool  tap  water, 
snow,  rain,  and  hail  gave  no  colon  bacilli. 

In  the  United  States  the  colon  test  has  been  exten- 
sively applied  during  the  last  few  years  to  certain  pol- 
luted river- waters,  in  particular  with  the  view  of  measuring 
the  purification  attained  by  sand  filtration  and  that 
naturally  occurring  during  the  flow  of  a  stream.  A  fairly 
good  idea  has  thus  been  obtained  of  the  numerical  dis- 
tribution of  the  B.  coli  in  the  larger  rivers.  Fuller  (Ful- 
ler. 1899),  for  example,  recorded  the  presence  of  colon 
bacilli  in  60  per  cent  of  the  i-c.c.  samples  taken  from  the 
Ohio  at  Cincinnati.  When  this  water  was  passed  through 


86  ELEMENTS  OF   WATER  BACTERIOLOGY. 

either  slow  sand  or  mechanical  niters,  the  effluent  gave 
positive  results  about  one-half  the  time  in  samples  of 
50  c.c. 

The  last  three  reports  of  the  Massachusetts  State 
Board  of  Health  contain  reports  of  the  abundance  of 
B.  coli  in  a  more  highly  polluted  stream,  the  Merrimac 
at  Lawrence.  In  1898  (Massachusetts  State  Board  of 
Health,  1899)  the  number  of  organisms  found  by  making 
litmus-lactose-agar  plates  directly  and  inspecting  the 
colonies  varied  from  20  per  c.c.  in  May  and  June  to  92  per 
c.c.  in  August  and  September,  the  average  for  the  year 
being  47.  Of  117  samples  of  the  water  which  had  passed 
through  the  city  filter,  only  9  showed  the  organism  in  a 
single  colony. 

In  1899  (Massachusetts  State  Board  of  Health,  1900) 
the  study  was  considerably  extended.  The  average  num- 
ber of  colon  bacilli  in  the  river  at  the  intake  of  the  filter 
was  again  47,  and  in  only  i  sample  out  of  180  was  it  absent ; 
below  the  city  at  the  Lawrence  Experiment  Station  the 
additional  pollution  raised  the  average  number  to  103. 
The  effluent  as  it  came  directly  from  the  filter  showed 
B.  coli  in  24  per  cent  of  the  cubic  centimeters  examined, 
but  at  the  outlet  of  the  reservoir,  the  proportion  had 
fallen  to  7  per  cent  and  at  the  Experiment  Station,  after 
passage  through  the  distribution  pipes,  to  only  4  per  cent. 
The  results  obtained  in  the  next  year,  1900,  were  prac- 
tically the  same,  but  parallel  tests  were  made  in  a  larger 
volume  of  water  by  incubating  with  the  addition  of  phenol 


THE  SIGNIFICANCE  OF  B.  COLI  IN  WATER.       87 

broth.     The  results  of  these  comparative  tests  may  be 
tabulated  as  follows: 

PERCENTAGE  OF  SAMPLES  OF  WATER  CONTAINING  B.  COLI. 

LAWRENCE    EXPERIMENT    STATION      (MASSACHUSETTS    STATE    BOARD     OF 
HEALTH,    IQOl). 

Effluent  of  Outlet  of  Tap,  Tap,  Experiment 

Filter.  Reservoir.  City  Hall.           Station. 

In  I  c.c 18.14  8.57  4.07                  1.87 

Iniooc.c 38.12  23.30  15.54              15.54 

It  appeared  that  the  use  of  the  larger  volume  of  water 
gave  very  little  additional  information,  and  indeed  the 
real  difference  between  the  waters  examined  is  rather 
obscured  by  the  use  of  the  large  samples. 

In  1900  Clark  and  Gage  (Clark  and  Gage,  1900)  re- 
ported some  specially  instructive  observations  made  when 
certain  of  the  underdrains  of  the  Lawrence  municipal 
water- filter  were  relaid  in  the  autumn  of  1898.  In  doing 
this  work  the  sand  on  some  of  the  beds  was  seriously 
disturbed;  and  in  December,  after  the  work  was  com- 
pleted, B.  coli  was  found  in  i  c.c.  of  the  filtered  efflu- 
ent in  72  per  cent  of  the  samples  examined.  In  Janu- 
ary and  February  the  organisms  were  found  in  54  per 
cent  and  62  per  cent  of  the  samples,  respectively,  while 
in  March  the  number  fell  to  a  normal  value  of  8  per  cent. 
Corresponding  to  this  excess  of  B.  coli  in  the  city  water, 
there  were  12  cases  of  typhoid  fever  in  December,  59  cases 
in  January,  12  in  February,  and  9  in  March,  all  during 
the  early  part  of  the  month.  The  authors  conclude  that 
"  when  filtering  a  river- water  as  polluted  as  that  of  the 


88  ELEMENTS  OF   WATER  BACTERIOLOGY. 

Merrimac,  it  is  safe  to  assume  that  when  B.  coli  is  found 
only  infrequently  in  i  c.c.  of  the  effluent  the  typhoid  germs, 
necessarily  fewer  in  number  and  more  easily  removed  by 
the  filter,  have  been  eliminated  from  the  water." 

Another  interesting  contribution  to  this  question  was 
made  by  the  Massachusetts  State  Board  of  Health 
(Massachusetts  State  Board  of  Health,  1901)  in  connec- 
tion with  the  examination  of  the  spring-waters  bottled 
for  sale  in  the  State.  Ninety-nine  springs  were  included 
in  this  study;  and  in  almost  every  instance  4  samples 
were  examined,  2  taken  directly  from  the  spring  by 
the  engineers  of  the  board  and  2  from  the  bottles  as 
delivered  for  sale  to  the  public.  In  the  water  of  one 
spring  B.  coli  was  found  twice,  once  in  a  sample  from 
the  spring  and  once  in  the  bottled  sample.  This  spring 
was  situated  in  woodland,  but  was  unprotected  from 
surface  drainage,  and  the  method  of  filling  bottles  sub- 
jected it  to  possible  contamination.  In  5  other  cases  B. 
coli  was  found  once  in  the  sample  from  the  spring;  all 
were  subject  to  pollution  from  dwellings  or  cultivated 
fields,  and  4  of  the  5  were  shown  to  be  highly  contam- 
inated, chemically.  In  7  other  cases  B.  coli  was  found 
in  the  bottled  samples  alone ;  3  of  these  sources  were  of 
high  purity,  but  the  bottling  process  furnished  opportunity 
for  contamination. 

Probably  the  most  elaborate  application  of  the  colon 
test  which  has  ever  been  attempted  was  made  by  Jordan 
in  his  recent  examinations  of  the  fate  of  the  Chicago  sew- 
age in  the  Desplaines  and  Illinois  Rivers.  At  one  time 


THE  SIGNIFICANCE  OF  B.  CO  LI  IN   WATER.       89 

Professor  Jordan  was  himself  somewhat  sceptical  as  to 
the  value  of  the  colon  test,  for  he  stated  in  1890  (Jordan, 
1890)  that  he  had  found,  "in  spring- water  which  was 
beyond  any  suspicion  of  contamination,  bacteria  which 
in  form,  size,  growth  on  gelatin,  potato,  etc.,  were  indis- 
tinguishable from  B.  coli  commune."  In  his  recent 
studies  of  self-purification  (Jordan,  1901)  the  analyses 
were  made  quantitative  by  the  examination  of  numerous 
measured  samples,  fractions  of  the  cubic  centimeter;  and 
the  method  employed  was  the  enrichment  either  in  dex- 
trose-broth fermentation- tubes  or  in  phenol  broth, 
with  the  subsequent  making  of  litmus-lactose-agar  plates. 
The  cultures  isolated  on  these  plates  were  tested  as  to 
their  behavior  in  dextrose  broth,  peptone  solution,  milk, 
and  gelatin;  of  the  dextrose  tubes  made  directly  from 
the  water  all  were  considered  positive  which  gave  more 
than  20  per  cent  gas  in  the  closed  arm,  with  an  appreciable 
excess  of  hydrogen.  The  results  were  very  significant. 
In  fresh  sewage  a  positive  result  was  obtained  about 
one-third  of  the  time  in  one  one-hundred-thousandth 
of  a  cubic  centimeter  and  almost  constantly  in  one  ten- 
thousandth  of  a  cubic  centimeter.  The  Illinois  and 
Michigan  canal  proved  almost  as  bad,  giving  positive 
results  on  seven  days  out  of  twenty-eight  in  dilution  of 
one  in  one  hundred  thousand  and  on  twenty-eight  days  out 
of  thirty-two  in  a  dilution  of  one  in  ten  thousand.  At 
Morris,  twenty-seven  miles  below  Lockport,  where  the 
canal  enters  the  bed  of  the  Desplaines  River,  and  nine 
miles  below  the  entrance  of  the  Kankakee,  the  principal 


9°  ELEMENTS  OF   WATER  BACTERIOLOGY. 

diluting  factor,  the  numbers  were  so  reduced  that 
positive  results  were  obtained  only  on  eleven  days  out 
of  twenty  in  one-thousandth  of  a  cubic  centimeter,  on 
twenty  days  out  of  thirty  in  one-hundredth  of  a  cubic 
centimeter,  and  on  twenty  days  out  of  twenty-three  in 
one-tenth  of  a  cubic  centimeter.  At  Averyville,  one  hun- 
dred and  fifty-nine  miles  below  Chicago,  colon  bacilli  were 
isolated  on  only  four  days  out  of  twenty-seven  in  one- 
tenth  of  a  cubic  centimeter,  and  on  thirteen  days  out  of 
thirty-one  in  one  cubic  centimeter.  A  comparison  with 
certain  neighboring  rivers  showed  this  to  be  about  the 
normal  value  for  waters  of  that  character,  as  the  following 
table  extracted  from  Professor  Jordan's  paper  will  show. 

NUMBER  OF  B.  COLI  PRESENT  IN  CERTAIN  RIVER  WATERS. 
(Jordan,  1901.) 

.1  c.c.  i  c.c. 

No.  Days        No.  Days     No.  Days       No.  Day 

Source  of  Sample.  Water  B.  Coli          Water  B.  Coli 

Examined.         Found.     Examined.         Found. 

Illinois  River,  Averyville 27  4  31  13 

Mississippi  River,  Grafton.  .       34  10  35  23 

Fox  River 22  2  23  6 

Sangamon  River 25  14  27  21 

Missouri  River 32  13  31  21 

These  results  harmonize  rather  closely  with  those  pre- 
viously recorded  by  Brown  and  Fuller  and  indicate  that 
in  the  larger  rivers  where  the  proportionate  pollution  is 
not  extreme,  colon  bacilli  may  be  isolated  in  about  half 
the  i -c.c.  samples  examined.  Such  rivers  are  of  course 
inadmissible  as  sources  of  water-supply,  according  to 
modern  sanitary  standards,  unless  subjected  to  pre- 
liminary treatment. 


THE  SIGNIFICANCE  OF  B.  COLI  IN   WATER.       91 

More  recently  Hunnewell  and  one  of  ourselves  (Wins- 
low  and  Hunnewell,  1902  b)  examined  a  considerable 
series  of  normal  waters  for  B.  coli,  testing  i  c.c. 
from  each  by  the  dextrose-broth  method  and  a  larger 
portion  of  100  c.c.  by  incubation  with  phenol  broth  as 
described  in  Chapter  VI.  The  samples  were  obtained 
from  the  public  supplies  of  Taunton,  Boston,  Cam- 
bridge, Braintree,  Brookline,  Needham,  and  Lynn  in 
Massachusetts,  and  of  Newport,  R.  L,  from  the  Sudbury 
River,  from  the  ocean,  from  the  waters  of  springs  bottled 
for  the  market,  from  ponds,  pools  of  rain  and  melted 
snow,  springs,  brooks,  shallow  wells,  and  driven  wells  in 
various  towns  near  the  city  of  Boston.  For  comparison 
50  samples  of  polluted  waters  from  the  Charles,  Mystic, 
Neponset,  and  North  Rivers  were  examined.  The  colon 
bacillus  was  defined  as  outlined  in  Chapter  VI;  and 
organisms  which  lacked  the  power  to  reduce  nitrates  or  to 
form  indol  were  classed  in  the  "Paracolon  group."  The 
results  are  summarized  in  the  following  table: 

PRESENCE  OF  B.  COLI  IN  POLLUTED  AND  UNPOLLUTED  WATERS. 

(Winslow  and  Hunnewell,  igo2b.) 
UNPOLLUTED   WATERS. 

I  C.C.  100  C.C. 

Samples  examined 157  153 

Dextrose  broth  positive 40  76 

Lactose  plate  positive 13  31 

Colon  group 5  n 

Paracolon  group 5  5 

B.  cloacae  group 5 

Streptococcus  group 3  10 


92  ELEMENTS  OF   WATER  BACTERIOLOGY. 

POLLUTED   WATERS. 

i  c.c.  100  c.c. 

Samples  examined 50  48 

Dextrose  broth  positive 50  37 

Lactose  plate  positive 50  26 

Colon  group 18  4 

Paracolon  group 6 

Streptococcus  group 25  22 

B.  cloacae i 

The  authors  pointed  out  that  these  tables  indicate 
that  bacteria  capable  of  growth  at  the  body  temperature 
and  fermenting  dextrose  and  lactose  are  infrequently  found 
in  unpolluted  waters,  and  colon  bacilli  are  very  rarely 
present^  since  in  157  samples  typical  colon  bacilli  only 
appeared  5  times.  In  the  polluted  waters  it  is  evident 
that  colon  bacilli  originally  present  were  in  many  cases 
killed  out  during  the  process  of  enrichment  by  the  strep- 
tococci, since  every  one  of  the  dextrose-broth  tubes  showed 
gas  at  the  first  incubation  of  i  c.c. 

The  latest  important  contribution  to  this  subject  conies 
from  Dantzic.  Petruschky  and  Pusch  (Petruschky  and 
Pusch,  1903)  examined  a  considerable  series  of  waters 
from  different  sources  by  incubating  measured  samples 
with  equal  amounts  of  nutrient  broth  and  isolating  upon 
agar.  In  45  samples  of  well-waters  they  found  B.  coli 
7  times  in  .01  c.c.,  9  times  in  .1  c.c.,  and  7  times  in  i  c.c. 
In  the  other  22  cases  it  could  not  be  found  in  i  c.c.  and  in 
4  cases  not  in  100  c.c.  One  sample  showed  it  only  in  .600 
c.c.  and  i  not  in  750  c.c.  Of  29  river- waters,  only  2  failed 
to  give  positive  results  in  .1  c.c.  and  14  showed  B.  coli  in 
,001  of  a  c.c.  or  less.  In  sewage  the  number  varied  from 


THE  SIGNIFICANCE  OF  B.  COLI  IN   WATER.       93 

i  to  1,000,000  per  c.c.  The  authors  conclude  that  a 
quantitative  estimation  of  the  B.  coli  content  furnishes  a 
good  measure  of  the  fecal  pollution  of  water  and  that 
the  observed  differences  in  the  extent  of  B.  coli  contam- 
ination of  surface-waters  are  so  great  that  they  differ 
from  each  other  more  than  a  million- fold. 

Altogether  the  evidence  is  quite  conclusive  that  the 
absence  of  B.  coli  demonstrates  the  harmlessness  of  a 
water  as  far  as  bacteriology  can  prove  it.  That  when 
present,  its  numbers  form  a  reasonably  close  index  of 
the  amount  of  pollution  the  authors  above  quoted 
have  proved  beyond  reasonable  cavil.  It  may  safely  be 
said  that  when  the  colon  bacillus,  as  denned  by  the  tests 
above,  is  found  in  such  abundance  as  to  be  isolated  in  a 
large  proportion  of  cases  from  i  c.c.  of  water,  it  is  reason- 
able proof  of  the  presence  of  serious  pollution,  a  view 
which  is  to-day  accepted  by  most  American  bacteriolo- 
gists. 


CHAPTER  VIII. 

PRESUMPTIVE  TESTS  FOR  B.  COLI. 

THE  isolation  and  identification  of  B.  coli  by  the 
methods  which  have  been  described  is  a  time-consuming 
and  laborious  operation,  and  one  sometimes  impossible 
to  apply  in  the  practical  supervision  of  a  water-supply. 

Hence  it  is  of  especial  value  to  the  engineer  to  have 
certain  tests  which  may  be  easily  and  quickly  carried  out 
and  which  will  give  a  probably  correct  idea  as  to  whether 
pollution  does  or  does  not  exist.  Such  tests  are  spoken  of 
as  "presumptive  tests."  If  positive,  the  water  showing 
such  a  test  may  be  regarded  as  suspicious,  but  must  be 
further  examined  to  prove  the  presence  of  fecal  bacteria. 
If  the  presumptive  test  is  negative,  no  further  examina- 
tion need  be  made.  Of  the  cultural  reactions  required 
to  establish  the  identity  of  the  colon  bacillus,  the  growth 
and  gas  formation  in  dextrose  broth,  as  originally  sug- 
gested by  Smith  (1893^  is  so  well  marked  and  so  strongly 
typical  of  B.  coli  that  this  medium  may  be  most  con- 
veniently used  as  a  "  presumptive  test." 

The  details  of  the  operation  vary  in  individual  labora- 
tories, but  the  underlying  principle  in  all  is  that  B.  coli 
develops  rapidly  in  dextrose  broth  with  gas  formation  of 

94 


PRESUMPTIVE   TESTS  FOR  B.  COLL  95 

from  25  to  70  per  cent  of  the  capacity  of  the  closed  arm 
of  the  fermentation- tube.  Of  this  gas  approximately 
one-third  is  carbon  dioxide  and  two-thirds  hydrogen, 

that  is,  as  the  gas  formula  is  generally  expressed,  7^-=  -• 

v^O2     i 

In  testing  a  water  by  this  method  a  series  of  samples 
of  the  water,  in  suitable  dilution,  .001,  .01,  .1,  i.o,  or  10  c.c., 
is  added  directly  to  the  dextrose-broth  tubes  and  incu- 
bated for  twenty- four  hours  at  37°. 

On  measurement  of  the  gas,  if  the  results  above  given 
are  obtained,  the  reaction  is  considered  typical.  If  the 
amount  of  gas  is  between  10  and  25  per  cent  or  more  than 
70  per  cent,  or  if  the  percentage  of  carbon  dioxide  is 
greater  than  40,  the  reaction  is  considered  atypical.  If 
no  gas  forms,  or  less  than  10  per  cent,  the  test  is  called 
negative.  It  is  recognized  that  the  distinction  is  not 
absolute,  since  even  pure  cultures  of  B.  coli  sometimes 
fail  to  give  typical  reactions,  and  on  the  other  hand,  an 
atypical  organism  sometimes  gives  a  satisfactory  positive 
test.  There  can  be  little  question,  however,  as  to  its 
value  to  the  sanitary  engineer. 

In  recent  years,  Irons  (Irons,  1901)  was  perhaps  the 
first  to  call  attention  to  the  value  of  this  method,  stating 
that  "when  the  dextrose  tube  yields  approximately 
33  per  cent  of  CO2  Bacillus  coli  communis  is  almost 
invariably  present."  In  the  next  year  the  reliability  of 
the  fermentation  test  as  an  indication  of  B.  coli  was 
worked  out  by  Gage  (Gage,  1902)  as  given  in  the  follow- 
ing table. 


96 


ELEMENTS  OF  WATER  BACTERIOLOGY. 


Number  of  samples  tested ^ 5T72 

"        giving  preliminary  fermentation.  .  .  .    1036 
Per  cent  of  latter  proved  to  contain  coli 70 


474 


The  work  of  Hunnewell  and  one  of  ourselves  (Wins- 
low  and  Hunnewell,  i902b)  showed  that  while  50  samples 
of  polluted  waters  all  gave  gas  in  the  dextrose  tube,  only 
40  out  of  157  samples  from  presumably  unpolluted  sources 
showed  a  similar  fermentation.  Whipple  (Whipple,  1903) 
examined  a  large  number  of  surface-water  supplies  by 
the  tl presumptive  test"  and  obtained  striking  results, 
shown  in  the  following  table.  The  waters  are  arranged 
in  six  groups  according  to  the  results  of  sanitary  inspec- 
tion, group  I  including  waters  collected  from  almost 
uninhabitated  watersheds  and  group  VI  waters  too  much 
polluted  to  be  safely  used  for  domestic  purposes. 

PERCENTAGE  OF  SAMPLES  OF  WATERS  OF  VARIOUS  SANITARY  GRADES 
GIVING  POSITIVE  TESTS  FOR  B.  COLI  WHEN  DIFFERENT  AMOUNTS 
WERE  EXAMINED. 

(Whipple,  1903.) 


Group. 

O.I  C.C. 

I.OC.C. 

10  C.C. 

IOO  C.C. 

500  c.c. 

I                                      

o.o 

».* 

20.8 

50.0 

CO.O 

II                          

s.o 

7.2 

IS.O 

60.0 

$v.v 
OO.  O 

HI                          

o.o 

7.0 

=;o.o 

so.o 

6o.O 

IV             '. 

4.0 

6.8 

41.7 

67.0 

7^.O 

v       

q.o 

13.0 

75.0 

IOO.O 

IOO.O 

VI      

5.0 

20.2 

75.0 

80.0 

IOO.O 

In  view  of  these  results  Whipple  suggests  the  following 
provisional  scheme  of  interpretation,  which  seems  to  the 
authors  to  be,  in  general,  sound. 


PRESUMPTIVE   TESTS  FOR  B.  COLL 


97 


Sanitary  Quality. 

Presumptive  Test  for  Bacillus  Coli. 

O.OI  C.C. 

O.I  C.C. 

I.O  C.C. 

IO.O  C.C. 

100  C.C. 

Safe.  .  . 

0 
0 
0 
0 

+ 

0 
0 
0 

+ 
+ 

0 
0 

+ 
+ 
+ 

o 

+ 
+ 
+ 
+ 

+ 
+ 
•f 
+ 
4- 

Reasonably  safe. 

Questionable  

Probably  unsafe 

Unsafe 

Results  harmonizing  well  with  the  above  were  obtained 
by  Nibecker  and  one  of  us  (Winslow  and  Nibecker,  1903), 
as  quoted  in  Chapter  IV.  Of  775  dextrose  tubes  inocu- 
lated from  259  samples  of  water  from  apparently  unpol- 
luted sources,  only  41  showed  gas  and  only  3  gave  the  gas 
formula  characteristic  of  B.  coli. 

The  litmus-lactose-agar  plate  furnishes  another  pre- 
sumptive test  of  considerable  value  as  indicated  in  Chap- 
ter IV,  although  it  is  probably  less  delicate  than  the  dex- 
trose-broth method.  With  polluted  waters  in  particular 
this  test  will  be  found  of  value,  since  streptococci  may 
interfere  with  the  dextrose-broth  method  if  dilution  is 
insufficient  or  incubation  too  prolonged. 

Certain  special  media  have  also  been  suggested  for 
rapid  routine  water  analysis  of  which  those  containing 
"  neutral  red,"  one  of  the  safranine  dyes,  has  been  most 
fully  studied.  Rothberger  (Rothberger,  1898)  first  pointed 
out  that  B.  coli  reduces  solutions  of  this  substance,  the 
color  changing  to  canary-yellow  accompanied  by  green 
fluorescence.  Makgill  (Makgill,  1901),  Savage  (Savage, 
1901),  and  other  English  observers  report  favorable 


98  ELEMENTS  OF  WATER  BACTERIOLOGY. 

results  from  the  use  of  this  test,  but  according  to  Ameri- 
can standards,  Irons  (Irons,  1902)  and  Gage  and  Phelps 
(Gage  and  Phelps,  1903)  conclude  that  the  group  of 
organisms  giving  a  positive  neutral  red  reaction  is  too 
large  a  one  to  give  very  valuable  sanitary  information. 
In  a  paper  read  at  the  Washington  meeting  of  the  Ameri- 
can Public  Health  Association  in  1903,  Stokes  suggested 
a  modification  of  this  medium  in  which  lactose  is  sub- 
stituted for  dextrose,  since  many  saprophytic  organisms 
will  attack  the  latter  and  not  the  former  sugar. 

The  medium  devised  by  MacConkey  (MacConkey, 
1900;  MacConkey,  1901;  MacConkey  and  Hill,  1901) 
containing  sodium  taurocholate  as  its  characteristic 
ingredient  has  also  proved  successful  in  the  hands  of 
some  observers.  The  dextrose  fermentation-tube,  how- 
ever, is  the  only  presumptive  test  whose  value  has  been 
established  by  conclusive  general  investigations. 


CHAPTER  IX. 

OTHER  INTESTINAL  BACTERIA. 

IT  would  be  an  obvious  advantage  if  the  evidence  of 
sewage  contamination,  furnished  by  the  presence  of  B.   o5 
coli  could  be  reinforced  or  confirmed  by  the  discovery  in   ^ 
water  of  other  forms  equally  characteristic  of  the  intestinal    2 
canal.     The  attention  of  a  few  bacteriologists  in  England    ^ 
and  America  has  been  turned  strongly  in  this  direction 
during  the  past  few  years;  and  two  groups  of  organisms, 
the  sewage  streptococci  and  the  anaerobic  spore-bearing    ^ 
bacilli,  have  been  described  as  being  probably  significant.     ^ 

The  term  "sewage  streptococci"  covers  an  ill-defined  ^ 
group  including  many  organisms  which  do  not  actually 
occur  in  well-marked  chains.  Those  most  commonly 
found  correspond,  however,  rather  closely  to  the  type  of 
Str.  erysipelatos.  They  grow  feebly  on  the  surface  of 
ordinary  nutrient  agar,  producing  faint  transparent, 
rounded  colonies,  but  under  semi-anaerobic  conditions 
flourish  better,  giving  a  well-marked  growth  along  the 
gelatin  stab  and  only  a  small  circumscribed  film  on  the 
surface.  They  are  favored  by  the  presence  of  the  sugars 
and  ferment  dextrose  and  lactose,  with  the  formation  of 
abundant  acid  but  no  gas.  They  are  seen  under  the 

99 


100         ELEMENTS  OF  WATER  BACTERIOLOGY. 

microscope  as  cocci,  occurring  as  a  rule  in  pairs,  short 
chains,  or  irregular  groups.  They  do  not  show  visible 
growth  in  the  standard  peptone  and  nitrate  solutions; 
most  of  them  do  not  liquefy  gelatin,  though  occasion- 
ally forms  are  found  which  possess  this  power.  No  sys- 
tematic study  of  the  various  species  found  in  the  intes- 
tine has  so  far  been  made,  and  at  present  all  cocci  giving 
the  characteristic  growth  on  agar  and  strongly  fermenting 
lactose  may  be  included  as  "  sewage  streptococci." 

Although  the  significance  of  the  streptococci  as  sewage 
organisms  is  not  established  with  the  same  definiteness 
which  marks  our  knowledge  of  the  colon  group,  these 
organisms  have  been  isolated  so  frequently  and  with  such 
constant  results  that  it  now  seems  reasonable  to  regard 
their  presence  as  indicative  of  pollution.  Although  orig- 
inally reported  by  Laws  and  Andrewes  (Laws  and 
Andre wes,  1894),  their  importance  was  not  emphasized 
until  1899  and  1900,  when  Houston  (Houston,  i899b, 
i9cob)  laid  special  stress  upon  the  fact  that  streptococci 
and  staphylococci  seem  to  be  characteristic  of  sewage  and 
animal  waste,  the  former  being,  in  his  opinion,  the  more 
truly  indicative  of  dangerous  pollution,  since  they  are 
"  readily  demonstrable  in  w-aters  recently  polluted  and 
seemingly  altogether  absent  from  waters  above  suspicion 
of  contamination."  In  the  water  of  6  rivers  recently 
extensively  sewage-polluted  he  found  streptococci  in 
from  one-tenth  to  one  ten-thousandth  of  a  c.c.  of  the  water 
examined,  although  in  some  cases  the  chemical  analysis 
did  not  clearly  indicate  dangerous  pollution.  On  the 


OTHER  INTESTINAL  BACTERIA.  IOI 

other  hand,  8  rivers,  polluted,  but  not  recently  and  exten- 
sively polluted,  showed  no  streptococci  in  one-tenth  of  a 
c.c.,  although  the  chemical  and  the  ordinary  bacteriological 
tests  gave  results  which  would  condemn  the  waters.  Hor- 
rocks  (Horrocks,  1901)  found  these  organisms  in  great 
abundance  in  sewage  and  in  waters  known  to  be  sewage- 
polluted,  but  which  contained  no  traces  of  Bacillus  coli. 
He  found  by  experiment  that  B.  coli  gradually  disappeared 
from  many  specimens  of  sewage  kept  in  the  dark  at  the 
temperature  of  an  outside  veranda,  while  the  commonest 
forms  which  persisted  were  varieties  of  streptococci  and 
staphylococci. 

In  America  attention  was  first  called  to  these  organisms 
by  Hunnewell  and  one  of  ourselves  (Winslow  and  Hunne- 
well,  1902*),  and  the  same  authors  later  (Winslow  and 
Hunnewell,  i9O2b)  recorded  the  isolation  of  streptococci 
from  25  out  of  50  samples  of  polluted  waters.  Gage 
(Gage,  1902),  from  the  Lawrence  Experiment  Station 
has  reported  the  organisms  present  in  the  sewage  of  that 
city,  while  one  of  us  (Prescott,  i902b)  has  shown  that 
they  are  abundant  in  fecal  matter  and  often  overgrow 
B.  coli  in  a  few  hours  when  inoculations  are  made  from 
such  material  into  dextrose  broth.  In  the  recent  mono- 
graph of  Le  Gros  (Le  Gros,  1902)  of  the  many  strep- 
tococci described  all  without  exception  were  isolated, 
either  from  the  body  or  from  sewage.  In  the  study  of 
259  samples  of  presumably  unpolluted  waters,  by  the 
method  of  direct  plating,  Nibecker  and  one  of  the  authors 
(Winslow  and  Nibecker,  1903)  found  streptococci  in 


102         ELEMENTS  OF  WATER  BACTERIOLOGY. 

only  i  sample,  while  Baker  and  one  of  us  (Prescott  and 
Baker,  1904)  found  the  organism  present  in  each  of 
50  samples  of  variously  polluted  waters  when  inoculation 
was  first  made  into  dextrose  broth  from  which  litmus- 
lactose-agar  plates  were  made  after  various  intervals. 

The  isolation  of  these  organisms  either  from  plates  or 
liquid  cultures  is  easy.  On  the  lactose-agar  plate,  made 
directly  from  a  polluted  water  the  colonies  of  the  strep- 
tococci may  generally  be  distinguished  from  those  of  other 
acid-formers  by  their  small  size,  compact  structure,  and 
deep- red  color,  which  is  permanent,  never  changing  to  blue 
at  a  later  period  of  incubation  as  the  colonies  of  B.  coli 
may  do.  Developing  somewhat  slowly,  however,  they 
may  be  overlooked  if  present  only  in  small  numbers.  In 
the  dextrose- broth  tube,  streptococci  will  appear  in  abun- 
dance after  a  suitable  period  of  incubation.  Prescott 
and  Baker  in  the  work  above  mentioned  found  that  with 
mixtures  of  B.  coli  and  streptococci  in  which  the  initial 
ratios  of  the  latter  to  the  former  varied  from  i :  94  to  208 :  i, 
the  colon  bacilli  developed  rapidly  during  the  early  part 
of  the  experiment,  reaching  a  maximum  after  about 
fourteen  hours  and  then  diminishing  rapidly,  while  the 
streptococci  first  became  apparent  after  ten  to  fifteen 
hours  and  reached  their  maximum  after  twenty  to  sixty 
hours,  according  to  the  number  originally  present. 

Applying  the  same  method  to  polluted  waters,  similar 
periodic  changes  were  observed;  pure  cultures  of  B.  coli 
were  first  obtained,  then  the  gradual  displacement  of  one 
form  by  the  other  took  place,  and  at  length  streptococci 


OTHER  INTESTINAL  BACTERIA. 


103 


were  either  present  in  pure  culture  or  in  great  predomi- 
nance as  shown  by  the  accompanying  tables.  The  samples 
of  water  were  plated  directly  upon  litmus-lactose-agar  and 
he  plates  were  incubated  at  37°  for  twenty-four  hours, 
when  the  red  colonies  were  counted.  At  the  time  of  plating, 
i  c.c.  from  each  sample  was  also  inoculated  into  dextrose 
broth  in  fermentation-tubes,  which  were  likewise  incu- 
bated at  37°.  After  various  periods,  as  indicated  by  the 
tables  below,  the  tubes  were  shaken  thoroughly  and  i  c.c. 
of  the  contents  withdrawn.  This  was  then  diluted 
(generally  1-1,000,000)  with  sterile  water,  plated  with 

TABLE  I. 

RELATIVE      GROWTH    OF      B.     COLI    AND    SEWAGE      STREPTOCOCCI    FROM 
POLLUTED   WATERS   IN   DEXTROSE    BROTH. 

(Prescott  and  Baker,  1904.)  • 


Sample  Number  

i 

2 

3 

4 

5 

6 

7 

8 

9 
1250 
420 

10 

105 
410 

Red  colonies  d 
from  i  c.c.  o 
sample  on  b 
tose-agar.  .  . 

eveloping  ) 
f  original  ( 
tmus-lac-  f 

4 

IO 

9 
68.4 

5 

8 

55 
400 

35 
13° 

460 

Number 
tound.  in 
millions 
per  cubic- 
centime- 
ter, after- 
growth in 
dextrose 
broth  for 
various 
periods.  . 

ii 

hrs. 

B.  coli 

0 

20 

200 

185 

332 

Strept. 

0 

0 

0 

0 

0 

0 

0 

o 

0 

0 

16 

hrs. 

& 

B.  coli 

200 
40 
280 
140 

76 

130 

20 

270 

10 

220 

45 

210 

3° 

140 

20 

420 

285 
75 

410 
145 

300 
350 

Strept. 

25 

210 

B.  coli 
Strept. 

15° 

85 

385 

370j    300 
170     300 

570 
1700 

2OO 

no 

4°5 

320 

280 

350 

370 

39 

hrs. 

B.  coli 

o 

0 

420 
o 

25 
480 

no!        o 

210 

20 

24 

105 

0 

170 

Strept. 

474 

300 

0 

45 

390 

170 

400 

105 

250 

£3 

hrs. 

B.  coli 

0 

0 

o 

i 

12 

2 

8 
45 

0 
ISO 

o 
86 

Street. 

2 

o 

O 

First  gas  noted  after  (hrs)  . 

10 

10 

9 

9 

10 

8 

1C 

6 

6 

8 

104         ELEMENTS  OF   WATER  BACTERIOLOGY. 

TABLE  II. 

RELATIVE    GROWTH      OF    B.     COLI    AND    SEWAGE      STREPTOCOCCI     FROM 

POLLUTED   WATERS    IN    DEXTROSE    BROTH. 

(Prescott  and  Baker,  1904.) 


Sample  Number  

T« 

19 

20 

21 

22 

23 

24 

25 

25 

30 

Red  colonies  developing  from  i  c.c.  ) 
of  original  sample  on  litmus-lac-  >• 

3° 

So 

170 

200 

tose-agar  i 

7 

B.  coli 

.02 

.01 

.04 

.12 

•  55 

1.6 

hrs. 

Strept 

IOO 

88 

350 

1  60 

B.  coli 

266 

Sio 

380 

330 

hrs. 

Strept. 

ISO 

0 

40 

140 

240 

128 

80 

220 

millions  per  cubic 

27 

B.  coli 

520 

610 

72 

700 

IOOO 

740 

IOO 

300 

growth  in  dextrose 

hrs. 

Strept. 

800 

860 

670 

1080 

2500 

4380 

3900 

40 

B.  coli 

o 

0 

10 

22 

36 

7 

7 

hrs. 

Strept. 

252 

330 

260 

22 

66 

60 

52 

B.  coli 

10 

16 

38 

20 

70 

35 

IO 

27 

• 

hrs. 

Strept. 

40 

16 

3-8 

31 

4i 

25 

10 

30 

litmus-lactose-agar  in  the  usual  way,  and  incubated  for 
twenty- four  hours.  The  colonies  of  B.  coli  and  strep- 
tococci were  distinguished  microscopically,  and  by  differ- 
ence in  color  and  general  characters. 

These  facts  suggest  the  following  method  for  the  detec- 
tion of  B.  coli  and  sewage  streptococci  in  polluted  waters. 

Inoculate  the  desired  quantity  of  water,  preferably  i  c.c., 
into  dextrose  broth,  in  a  fermentation-tube,  and  incubate 
at  37°.  After  a  few  hours'  incubation  examine  the  cul- 
tures for  gas.  Within  two  or  three  hours  after  gas  for- 
mation is  first  evident,  plate  from  the  broth  in  litmus- 
lactose-agar,  incubating  for  twelve  to  eighteen  hours  at 


OTHER  INTESTINAL  BACTERIA.  105 

37°.  If  at  the  end  of  this  time  no  acid-producing  colonies 
are  found,  it  is  probably  safe  to  assume  that  there  were  no 
colon  bacilli  present.  On  the  other  hand,  if  red  colonies 
are  developed,  these  must  be  further  examined  by  the 
regular  diagnostic  tests  for  B.  coli.  After  the  first  plating 
from  the  dextrose  broth,  replace  the  fermentation-tube 
in  the  incubator  and  allow  it  to  remain  for  twenty-four 
to  thirty-six  hours,  then  plate  again  in  litmus-lactose-agar. 
This  plating  should  give  a  nearly  pure  culture  of  strep- 
tococci if  these  organisms  were  originally  present  in  the 
water.  If  colon  bacilli  are  not  found  in  the  first  set  of 
plates  the  streptococci  may  still  be  isolated  by  this  method. 
The  relative  relation  of  the  streptococci  and  the  colon 
bacilli  to  sewage  pollution  is  still  uncertain.  Houston 
(Houston,  1900)  held  that  the  former  microbes  imply 
"  animal  pollution  of  extremely  recent  and  therefore 
specially  dangerous  kind."  Horrocks  (Horrocks,  1901), 
on  the  other  hand,  maintains,  largely  on  the  strength  of 
certain  experiments  with  stored  sewage,  that  the  strep- 
tococci persist  after  colon  bacilli  have  disappeared  and 
indicate  contamination  with  old  sewage  which  is  not 
necessarily  dangerous.  On  the  whole,  it  seems  probable 
that  the  streptococci,  like  the  colon  bacilli,  are  primarily 
parasitic  organisms,  the  former  being  associated  both 
with  the  outer  and  inner  surfaces  of  the  body.  Like  the ' 
colon  bacilli,  their  presence  in  water  indicates  contact 
with  the  wastes  of  human  life,  and  their  isolation  from 
a  suspected  sample  furnishes  valuable  confirmatory  evi- 
dence of  its  dangerous  character. 


106          ELEMENTS  OF   WATER  BACTERIOLOGY. 

The  English  bacteriologists  have  ascribed  much  impor- 
tance as  indicators  of  sewage  pollution  to  another  group 
of  organisms,  the  anaerobic  spore-forming  bacilli,  of  which 
the  B.  sporogenes  is  a  type.  This  form  was  isolated  by 
Klein  (Klein,  1898;  Klein,  1899)  in  1895,  in  the  course 
of  an  epidemic  of  diarrhoea  at  St.  Bartholomew's  Hos- 
pital, and  described  under  the  name  of  B.  enteritidis 
sporogenes;  it  is  apparently  identical  with  the  B.  aero- 
genes  capsulatus  of  Welch  (Welch  and  Nuttall,  1892). 

Klein's  procedure  for  isolating  the  B.  sporogenes  is 
simple  in  the  case  of  polluted  waters.  A  portion  of  the 
sample  to  be  examined  is  added  to  a  tube  of  sterile  milk, 
which  is  then  heated  to  80°  C.  for  ten  minutes  to  destroy 
vegetative  cells.  The  milk  is  then  cooled  and  incubated 
under  anaerobic  conditions,  which  may  be  accomplished 
most  conveniently  by  Wright's  method.  A  tight  plug  of 
cotton  is  forced  a  quarter  way  down  the  test-tube,  the 
space  above  is  loosely  filled  with  pyrogallic  acid,  a  few 
drops  of  a  strong  solution  of  caustic  potash  are  added, 
and  the  tube  is  tightly  closed  with  a  rubber  stopper.  After 
eighteen  to  thirty-six  hours  at  37°  the  appearance  of  the 
tube  will  be  very  characteristic  if  the  B.  sporogenes  is 
present.  "The  cream  is  torn  or  altogether  dissociated 
by  the  development  of  gas,  so  that  the  surface  of  the 
medium  is  covered  with  stringy,  pinkish-white  masses  of 
coagulated  casein,  enclosing  a  number  of  gas-bubbles. 
The  main  portion  of  the  tube  formerly  occupied  by  the 
milk  now  contains  a  colorless,  thin,  watery  whey,  with  a 
few  casein  lumps  adhering  here  and  there  to  the  sides  of 


OTHER  INTESTINAL  BACTERIA  107 

the  tube.  When  the  tube  is  opened,  the  whey  has  a  smell 
of  butyric  acid  and  is  acid  in  reaction.  Under  the  micro- 
scope the  whey  is  found  to  contain  numerous  rods,  some 
motile,  others  motionless." 

The  B.  sporogenes  when  isolated  in  pure  culture  on 
glucose  agar  is  a  stout  rod.  It  liquefies  gelatin,  forming 
in  this  medium  large  oval  spores.  It  is  strongly  patho- 
genic for  guinea-pigs,  by  which  character  it  is  distinguished 
from  the  B.  butyricus  of  Botkin. 

The  researches  of  Klein  and  Houston  (Klein  and  Hous- 
ton, 1898,  1899)  have  shown  that  the  B.  sporogenes  occurs 
in  English  sewage  in  numbers  varying  from  30  to  2000  per 
c.c.  and  that  it  is  often  absent  in  considerable  volumes  of 
pure  water.  In  Boston  sewage  it  may  usually  be  isolated 
from  .01  or  .001  of  a  c.c.  (Winslow  and  Belcher,  1904). 

Evidently  in  order  to  have  any  significance,  an  exam- 
ination for  this  organism  must  be  made  with  large  samples 
and  the  concentration  of  at  least  2000  c.c.  of  water  through 
a  Pasteur  filter  is  recommended  by  Horrocks  as  a  neces- 
sary prelude  (Horrocks,  1901).  Since  the  spores  of  an 
anaerobic  bacillus  may  persist  for  an  indefinite  period  in 
polluted  waters,  their  presence  need  not  necessarily  indi- 
cate recent  or  dangerous  pollution.  Since  the  number 
present  even  in  sewage  is  so  small  and  so  variable,  no 
quantitative  standard  can  be  established;  on  the  whole, 
it  does  not  appear  that  the  practical  application  of  the 
anaerobic  test  will  ever  be  a  wide  one. 

There  are  numerous  other  sewage  bacteria  whose 
presence  is  more  or  less  constantly  characteristic  of  pol- 


108         ELEMENTS  OF  WATER  BACTERIOLOGY. 

luted  waters.  Organisms  of  the  Proteus  group  are  some- 
times present,  exhibiting  marked  morphological  varia- 
tions, from  the  coccus  form  to  long  twisted  threads  and 
forming  on  gelatin  irregular  amoeboid  colonies  with  filiform 
processes  extending  into  the  surrounding  gelatin.  The 
B.  subtilis  group  of  strongly  aerobic  spore-forming  bacilli, 
giving  a  brown  wrinkled  parchment-like  growth  on  agar, 
and  moss-like  liquefying  colonies  on  gelatin,  is  usually 
represented.  Among  the  allies  of  B.  coli  may  be  men- 
tioned B.  aerogenes,  which  differs  from  it  in  being  non- 
motile,  failing  to  produce  indol,  and  forming  spherical 
drop-like  colonies  on  gelatin.  B.  cloacae  resembles  B.  coli 
in  most  respects,  but  causes  a  liquefaction  of  gelatin. 
The  property  of  liquefaction  was  formerly  believed  to  be 
of  general  significance,  inasmuch  as  the  liquefying  bacteria 
were  regarded  as  closely  allied  to  intestinal  organisms, 
and  in  themselves  indicative  of  pollution.  This  position 
is,  however,  no  longer  tenable,  since  many  bacteria, 
typical  of  the  purest  waters,  may  cause  liquefaction. 

While  the  organisms  above  mentioned,  and  many  others 
as  well,  deserve  notice  in  the  examination  of  gelatin  plates 
from  a  suspected  water,  none  of  them  is  of  sufficient 
importance  to  warrant  any  special  procedure  for  their 
isolation. 


CHAPTER  X. 

THE  SIGNIFICANCE  AND  APPLICABILITY  OF  THE  BAC- 
TERIOLOGICAL EXAMINATION. 

THE  first  attempt  of  the  expert  called  in  to  pronounce 
upon  the  character  of  a  potable  water  should  be  to  make 
a  thorough  sanitary  inspection  of  the  pond,  stream,  or  well 
from  which  it  is  derived.  Study  of  the  possible  sources 
of  pollution  on  a  watershed,  of  the  direction  and  velocity 
of  currents  above  and  below  ground,  of  the  character  of 
soil  and  the  liability  to  contamination  by  surface-wash 
are  conceded  to  yield  evidence  of  the  greatest  value. 
Often,  however,  some  opinion  must  be  formed  upon  the 
quality  of  water  sent  from  a  distance  without  the  oppor- 
tunity of  examining  its  surroundings;  and  even  when 
sanitary  inspection  can  be  made,  its  results  are  by  no 
means  conclusive.  No  reconnoissance  can  show  cer- 
tainly whether  unpurified  drainage  from  a  cesspool  does 
or  does  not  reach  a  given  well;  whether  sewage  discharged 
into  a  lake  does  or  does  not  find  its  way  to  a  neighboring 
intake;  whether  pollution  of  a  stream  has  or  has  not  been 
removed  by  a  certain  period  of  flow.  Evidence  upon 

these  points  must  be  obtained  from  a  careful  study  of  the 

109 


no         ELEMENTS  OF  WATER  BACTERIOLOGY. 

characteristics  of  the  water  in  question,  and  this  study 
can  be  carried  out  along  two  lines,  chemical  and  bac- 
teriological. 

A  chemical  examination  of  water  for  sanitary  purposes, 
is  mainly  useful  in  throwing  light  upon  one  point — the 
amount  of  decomposing  organic  matter  present.  Humus- 
like  substances  may  be  abundant  in  surface-waters  quite 
free  from  harmful  pollution,  but  these  are  stable  com- 
pounds. Easily  decomposable  bodies,  on  the  other  hand, 
must  obviously  have  been  recently  introduced  into  the 
water  and  mark  a  transitional  state.  "The  state  of 
change  is  the  state  of  danger,"  as  Dr.  T.  M.  Drown  has 
phrased  it.  Sometimes  the  organic  matter  has  been 
washed  in  by  rain  from  the  surface  of  the  ground,  some- 
times it  has  been  introduced  in  the  more  concentrated 
form  of  sewage.  In  any  case,  it  is  a  warning  of  possible 
pollution,  and  the  determination  of  free  ammonia,  nitrites, 
carbonaceous  matter,  as  shown  by  "  oxygen  consumed," 
and  dissolved  oxygen  yield  important  evidence  as  to  the 
sanitary  quality  of  a  water. 

Furthermore,  nitrates,  the  final  products  of  the  oxida- 
tion of  organic  matter,  and  the  chlorine  introduced  as 
common  salt  into  all  water  which  has  been  in  contact 
with  the  wastes  of  human  life,  furnish  additional  informa- 
tion as  to  the  antecedents  of  a  sample.  The  results  of 
the  chlorine  determination  are  indeed  perhaps  more  clear 
than  thfcse  of  any  other  sanitary  analysis,  for  chlorine 
and  sewage  pollution  vary  together,  due  allowance  being 
made  for  the  proximity  of  the  sea  and  other  geological 


BACTERIOLOGICAL  EXAMINATION.  ill 

and  meteorological  factors.  Unfortunately,  it  is  only 
past  history  and  not  present  conditions  which  these  latter, 
tests  reveal,  for  in  a  ground-water  completely  purified 
from  a  sanitary  standpoint  such  soluble  constituents 
remain,  of  course,  unchanged.  Thus,  in  the  last  resort, 
it  is  upon  the  presence  and  amount  of  decomposing 
organic  matter  in  the  water  studied  that  the  opinion  of 
the  chemist  must  be  based. 

The  decomposition  of  organic  matter  may  be  measured 
either  by  the  material  decomposed  or  by  the  number  of 
organisms  engaged  in  carrying  out  the  process  of  decom- 
position. The  latter  method  has  the  advantage  of  far 
greater  delicacy,  since  the  bacteria  respond  by  enormous 
multiplication  to  very  slight  increase  in  their  food-supply, 
and  thus  it  comes  about  that  the  standard  gelatin-plate 
count  at  20°  roughly  corresponds,  in  not  too  heavily 
polluted  waters,  to  the  free  ammonia  and  "oxygen  con- 
sumed," as  revealed  by  chemical  analysis.  If  low  num- 
bers of  bacteria  are  found,  the  evidence  is  highly  reassuring, 
for  it  is  seldom  that  water  could  be  contaminated  under 
natural  conditions  without  the  direct  addition  of  foreign 
bacteria  or  of  organic  matter  which  would  condition  a 
rapid  multiplication  of  those  already  present.  The  bac- 
teriologist in  such  cases  can  declare  the  innocence  of 
the  water  with  justifiable  certainty.  When  high  num- 
bers are  found  the  interpretation  is  less  simple,  since  they 
may  exceptionally  be  due  to  the  multiplication  of  certain 
peculiar  water  forms.  Large  counts,  however,  under 
ordinary  conditions,  when  including  a  normal  variety  of 


112         ELEMENTS  OF  WATER  BACTERIOLOGY. 

forms  indicate  the  presence  of  an  excess  of  organic  matter 
derived  in  all  probability  either  from  sewage  or  from  the 
fresh  washings  of  the  surface  of  the  ground.  In  either 
case  danger  is  indicated. 

A  still  closer  measure  of  polluting  material  may  be 
obtained  from  the  numbers  of  colonies  which  develop 
on  litmus-lactose-agar  at  37°,  since  organisms  which 
thrive  at  the  body  temperature,  and  particularly  those 
which  ferment  lactose,  are  characteristic  of  the  intestinal 
tract  and  but  rarely  occur  in  normal  waters. 

Finally,  the  search  for  the  Bacillus  coli  furnishes  the 
most  satisfactory  of  all  single  tests  for  fecal  contamination. 
This  organism  is  pre-eminently  a  denizen  of  the  alimen- 
tary canal  and  may  be  isolated  with  ease  from  waters  to 
which  even  a  small  proportion  of  sewage  has  been  added. 
On  the  other  hand,  it  is  never  found  in  abundance  in 
waters  of  good  sanitary  quality;  and  its  numbers  form 
an  excellent  index  of  the  value  of  waters  of  an  interme- 
diate grade.  The  streptococci  appear  to  be  forms  of  a 
similar  significance  useful  as  yielding  a  certain  amount  of 
confirmatory  evidence.  The  full  bacteriological  analysis 
should  then  consist  of  three  parts — the  gelatin-plate  count, 
as  an  estimate  of  the  amount  of  organic  decomposition 
in  process;  the  total  count,  and  the  count  of  red  colonies, 
on  litmus-lactose-agar,  as  a  measure  of  the  organisms 
which  form  acids  and  thrive  at  the  body  temperature; 
and  the  study  of  a  series  of  dextrose-broth  tubes  for  the 
isolation  of  colon  bacilli  and  streptococci.  The  simple 
examination  of  the  dextrose-broth  tube  and  the  count  on 


BACTERIOLOGICAL  EXAMINATION.  113 

the  litmus-lactose-agar  plate  serve  for  what  Whipple  has 
well  called  presumptive  tests. 

The  results  of  the  bacteriological  examination  have,  in 
several  respects,  a  peculiar  and  unique  significance. 
First,  this  examination  is  the  most  direct  method  of  sani- 
tary water  analysis.  What  we  dread  in  drinking-water  is 
the  presence  of  pathogenic  bacteria,  mainly  from  the 
intestinal  tract  of  man,  and  it  is  quite  certain  that  the 
related  non-pathogenic  bacteria  from  the  same  source 
will  behave  more  nearly  as  these  disease  germs  do  than 
will  any  chemical  compounds.  In  the  second  place,  the 
bacteriological  methods  are  superior  in  delicacy  to  any 
others.  Klein  and  Houston  (Klein  and  Houston,  1898) 
showed  by  experiment  with  dilutions  of  sewage  that  the 
colon  test  was  from  ten  to  one  hundred  times  as  sensitive 
as  the  methods  of  chemical  analysis;  and  studies  of  the 
self-purification  of  streams  have  confirmed  his  results 
on  a  practical  scale.  Thus  in  the  Sudbury  River  it  was 
found  that  while  the  chemical  evidences  of  pollution 
persisted  for  six  miles  beyond  the  point  of  entrance, 
the  bacteria  introduced  could  be  detected  for  four  miles 
further  (Woodman,  Winslow,  and  Hansen,  1902). 

The  statement  is  sometimes  made  that  while  bac- 
teriological methods  may  be  more  delicate  for  the  detec- 
tion of  pollution  in  surface-waters,  contamination  in 
ground-waters  may  best  be  discovered  by  the  chemical 
analysis.  That  such  is  not  the  case  has  been  well  shown 
by  Whipple  (Whipple,  1903),  who  cites  the  following 


114         ELEMENTS  OF  WATER  BACTERIOLOGY 

two  instances  in  which  the  presumptive  test  revealed  con- 
tamination not  shown  by  the  chemical  analysis: 

"A  certain  driven-well  station  was  located  in  swampy 
land  along  the  shores  of  a  stream,  and  the  tops  of  the 
wells  were  so  placed  that  they  were  occasionally  flooded 
at  times  of  high  water.  The  water  in  the  stream  was 
objectionable  from  the  sanitary  standpoint.  The  wells 
themselves  were  more  than  100  feet  deep;  they  pene- 
trated a  clay  bed  and  yielded  what  may  be  termed  arte- 
sian water.  Tests  for  the  presence  of  Bacillus  coli  had 
invariably  given  negative  results,  as  might  be  naturally 
expected.  Suddenly,  however,  the  tests  became  positive 
and  so  continued  for  several  days.  On  investigation  it 
was  found  that  some  of  the  wells  had  been  taken  up  to 
be  cleaned,  and  that  the  workmen  in  resinking  them  had 
used  the  water  of  the  brook  for  washing  them  down. 
This  allowed  some  of  the  brook- water  to  enter  the  system. 
It  was  also  found  that  at  the  same  time  the  water  in  the 
brook  had  been  high,  and  because  of  the  lack  of  packing 
in  certain  joints  at  the  top  of  the  wells  the  brook- water 
leaked  into  the  suction  main.  The  remedy  was  obvious 
and  was  immediately  applied,  after  which  the  tests  for 
Bacillus  coli  once  more  became  negative.  During  all 
this  time  the  chemical  analysis  of  the  water  was  not  suffi- 
ciently abnormal  to  attract  attention.  On  another  occa- 
sion a  water-supply  taken  from  a  small  pond  fed  by 
springs,  and  which  was  practically  a  large  open  well, 
began  to  give  positive  tests  for  Bacillus  coli,  and  on  exam- 
ination it  was  found  that  a  gate  which  kept  out  the  water 


BACTERIOLOGICAL  EXAMINATION.  11$ 

of  a  brook  which  had  been  formerly  connected  with  the 
pond  was  open  at  the  bottom,  although  it  was  supposed 
to  have  been  shut,  thus  admitting  a  contaminated  sur- 
face-water to  the  supply."  Whipple  also  calls  attention 
to  the  report  on  the  Chemical  and  Bacteriological  Exami- 
nation of  Chichester  Well-waters  by  Houston  (Houston, 
1901),  in  which  the  results  of  chemical  and  bacteriologi- 
cal examinations  of  30  wells  were  compared.  It  was 
found  that  the  bacteriological  results  were  in  general 
concordant  and  satisfactory.  The  wells  which  were  high- 
est in  the  number  of  bacteria  showed  also  the  greatest 
amount  of  pollution,  as  indicated  by  the  numbers  of  B. 
coli,  B.  sporogenes,  and  streptococci.  On  the  other 
hand,  the  chlorine  and  the  albuminoid  ammonia  showed 
no  correspondence  with  the  bacteriological  results. 

Thirdly,  negative  tests  for  Bacillus  coli  and  low  bac- 
terial counts  may  be  interpreted  as  proofs  of  the  good 
quality  of  water,  with  a  certainty  not  attainable  by  any 
other  method  of  analysis.  Many  a  surface-water  with 
reasonably  low  chlorine  and  ammonias  has  caused  epi- 
demics of  typhoid  fever;  but  it  is  impossible  under  any 
natural  conditions  that  a  water  could  contain  the  typhoid 
bacillus  without  giving  clear  evidence  of  pollution  in  the 
dextrose-broth  tube  or  on  the  lactose-agar  plate. 

It  seems  to  the  writers  that  the  real  application  of 
chemistry  begins  where  that  of  bacteriology  ends.  When 
pollution  is  so  gross  that  its  existence  is  obvious  and  only 
its  amount  needs  to  be  determined,  the  bacteriological 
tests  will  not  serve,  on  account  of  their  excessive  delicacy. 


n6         ELEMENTS  OP  WATER  BACTERIOLOGY. 

In  studying  the  heavy  pollution  of  small  streams,  the 
treatment  of  trades  wastes,  and  the  purification  of  sewage, 
the  relations  of  nitrogeneous  compounds  and  of  oxygen 
compounds  are  of  prime  importance.  In  other  words, 
when  pollution  is  to  be  avoided,  because  the  decompo- 
sition of  chemical  substances  causes  a  nuisance,  it  must 
be  studied  by  chemical  methods.  When  the  danger  is 
sanitary  and  comes  only  from  the  presence  of  bacteria, 
bacteriological  methods  furnish  the  true  index  of  pollution. 
In  the  study  of  certain  special  problems  the  paramount 
importance  of  bacteriology  is  generally  recognized.  The 
distribution  of  sewage  in  large  bodies  of  water  into  which 
it  has  been  discharged  may  thus  best  be  traced  on  account 
of  the  ready  response  of  the  bacterial  counts  to  slight 
proportions  of  sewage,  particularly  since  the  ease  and 
rapidity  with  which  the  technique  of  plating  can  be  carried 
out  make  it  possible  to  examine  a  large  series  of  samples 
with  a  minimum  of  time  and  trouble.  The  course  of  the 
sewage  carried  out  by  the  tide  from  the  outlet  of  the 
South  Metropolitan  District  of  Boston  was  studied  in 
this  way  by  E.  P.  Osgood  in  1897,  and  mapped  out  by 
its  high  bacterial  content  with  greater  accuracy  than 
could  be  attained  by  any  other  method.  Some  very 
remarkable  facts  have  been  developed  by  similar  studies 
as  to  the  persistence  of  separate  streams  of  water  in 
immediate  contact  with  each  other.  Heider  showed 
that  the  sewage  of  Vienna,  after  its  discharge  into  the 
Danube  River,  flowed  along  the  right  bank  of  the 
stream,  preserving  its  own  bacterial  characteristics,  and 


BACTERIOLOGICAL  EXAMINATION.  n? 

not  mixing  perfectly  with  the  water  of  the  river  for  a  dis- 
tance of  more  than  twenty-four  miles  (Heider,  1893). 
Jordan  (Jordan,  1900),  in  studying  the  self-purification  of 
the  sewage  discharged  from  the  great  Chicago  drainage 
canal,  found  by  bacteriological  analyses  that  the  Des 
Plaines  and  the  Kankakee  Rivers  could  both  be  distin- 
guished flowing  along  in  the  bed  of  the  Illinois,  the  two 
streams  being  in  contact,  yet  each  maintaining  its  own 
individuality.  Finally,  the  quickness  with  which  slight 
changes  in  the  character  of  a  water  are  marked  by 
fluctuations  in  bacterial  numbers  renders  the  bacterio- 
logical methods  invaluable  for  the  daily  supervision  of 
surface  supplies  or  of  the  effluents  from  municipal  nitra- 
tion plants. 

In  the  commoner  case,  when  normal  values  obtained 
by  such  routine  analyses  are  not  at  hand,  the  problem  of 
the  interpretation  of  any  sanitary  analysis  is  a  more  diffi- 
cult one.  The  conditions  which  surround  a  source  of 
water-supply  may  be  constantly  changing.  No  engineer 
can  measure  the  flow  of  a  stream  in  July  and  deduce  the 
amount  of  water  which  will  pass  in  February;  yet  the 
July  gauging  has  its  own  value  and  significance.  So  a 
single  analysis  of  any  sort  is  not  sufficient  for  all  past  and 
future  time.  If  it  gives  a  correct  picture  of  the  hygienic 
condition  of  the  water  at  the  moment  of  examination  it 
has  fulfilled  its  task,  and  this  the  bacteriological  analysis 
can  do.  The  evidence  furnished  by  inspection  and  by 
chemical  analysis  should  be  sought  for  and  welcomed 
whenever  it  can  be  obtained,  yet  we  are  of  the  opinion 


Ii8         ELEMENTS  OF  WATER  BACTERIOLOGY. 

that,  on  account  of  their  directness,  their  delicacy,  and 
their  certainty,  the  bacteriological  methods  should  least 
of  all  be  omitted,  and,  if  necessary,  they  alone  may  fur- 
nish conclusive  testimony  as  to  the  safety  of  a  potable 
water. 


APPENDIX. 


IT  has  been  pointed  out  that  the  number  of  bacteria 
developing  from  a  given  sample  of  water  will  be 
largely  dependent  upon  the  composition  and  character 
of  the  culture  medium.  As  the  nature  of  the  medium 
varies  widely  according  to  the  method  of  preparation, 
workers  in  different  laboratories  can  have  no  rational 
basis  for  comparison  of  results  until  their  methods  are 
essentially  uniform. 

To  make  results  comparable  as  far  as  possible,  the 
Laboratory  Section  of  the  American  Public  Health  Asso- 
ciation has  adopted  standard  methods  for  the  preparation 
of  the  commonly  used  nutrient  media  given  in  the  follow- 
ing extract  from  the  report  of  the  Committee  (Fuller,  1902). 

STANDARD  METHODS  FOR  GELATIN  AND  AGAR. 

GELATIN.  AGAR. 

I.  Boil   15  gm.  thread   agar  in   500  c.c. 

water  for  half  an  hour  and  make  up 
weight  to  500  gin.  or  digest  for  10 
minutes  in  the  autoclavat  110°  C. 
Let  this  cool  to  about  60°  C. 

a.  Infuse  500  gm.  lean  meat  24  hours  Infuse  500  gm.  lean  meat  24  hours 
with  1000  c.c.  of  distilled  water  with  500  c.c.  of  distilled  water  in  re- 
in refrigerator.  frigerator. 

3.  Make  up  any  loss  by  evaporation. 

4.  Strain  infusion  through  cotton  flannel. 

5.  Weigh  filtered  infusion. 

119 


120  APPENDIX. 

GELATIN.  A  GAR. 

6.  Add  i  %  Witte's  peptone  and  10%        Add  2  %  of  Witte's  peptone. 

gold  label  sheet  gelatin. 

7.  Warm  on  water-bath,  stirring  till  peptone  and  gela- 
tin are   dissolved  and  not  allowing  the  temperature  to 
rise  above  60°  C. 

8.  Neutralize. 

9.  To    500  gm.  of    the    meat    infusion 

add  500  c  c,  of  the  3%  agar,  keeping 
the  temperature  below  60°  C. 

10.  Heat  over  boiling  water  (or  steam)  bath  30  minutes. 

11.  Restore  loss  by  evaporation. 

12.  Titrate,  after  boiling  i  minute  to  expel  carbonic  acid. 

13.  Ad.iust  reaction  to  +1.0%  by  adding  normal  hydro- 
chloric acid  or  sodium  hydrate  as  required. 

14.  Boil  2  minutes  over  free  flame,  constantly  stirring. 

15.  Make  up  loss  by  evaporation. 

1 6.  Filter  through  absorbent  cotton  and  cotton  flannel, 
passing  the  filtrate  through  the  filter  until  clear. 

17.  Titrate  and  record  the  final  reaction. 

1 8.  Tube,  using  5  c.c.  in  each  tube  in  the  case  of  gelatin, 
and  7  c.c.  in  the  case  of  agar. 

19.  Sterilize  15  minutes  in  the  autoclav   at   no0,  or  for 
30  minutes  in  streaming  steam  on  three  successive  days. 

20.  Store  in  the  ice-chest  in  a  moist  atmosphere  to  pre- 
vent evaporation. 

Hill  (Hill,  1899)  has  arranged  the  methods  for  prepa- 
ration of  broth,  nutrient  gelatin,  and  nutrient  agar  in 
tabular  form  as  given  on  the  following  page. 

For  titration,  the  following  method,  suggested  by 
Fuller  (Fuller,  1895),  was  adopted  by  the  Public  Health 
Association  Committee  in  1897  (Committee  of  Bacteriolo- 
gists, 1898). 

The  medium  to  be  tested,  all  ingredients  being  dis- 
solved, is  brought  to  the  prescribed  volume  by  the  addition 
of  distilled  water  to  replace  that  lost  by  boiling,  and  after 
being  thoroughly  stirred,  5  c.c.  are  transferred  to  a  6-inch 
porcelain  evaporating-dish ;  to  this  45  c.c.  of  distilled  water 
are  added,  and  the  50  c.c.  of  fluid  are  boiled  for  three 
minutes  over  a  flame.  One  c.c.  of  a  .5  per  cent  solution 
of  phenolphthalein  in  50  per  cent  alcohol  is  then  added 

N 
and  the  reaction  is  determined  by  titration  with  —  sodium 


APPENDIX. 


121 


TABLE  SHOWING  ANALOGY  BETWEEN  BROTH,  NUTRIENT  GELATIN,  AND 
NUTRIENT  AGAR  MADE  BY  METHODS  HEREIN  RECOMMENDED. 


in 


Boil  30  gm.  thread  agar 
i  liter  of  water  for  half 
-^  hour.  Make  up  to  a 
weight  of  1000  gm. 

Cool  and  solidify. 


NUTRIENT  BROTH. 


NUTRIENT  GELATIN. 


NUTRIENT  AGAR. 


1.  Infuse     lean     meat     20 
hours  with  twice    its  weight 
of  distilled  water  in  refrig- 
erator. 

Say  1000  gm.  meat. 
'     2000    "     water. 

2.  Make     up     weight     of 
meat    infusion    (and    meat) 
to  original  weight  by  add- 
ing water,  i.e.  to  3000  gm. 

3.  Filter  infusion  through 
cloth  to  remove  meat. 

4.  Titrate   and   record   re- 
action  of   nitrate.     Say 
action  +  2.  2%. 


5.  Weigh    infusion. 
1800  gm. 


Say 


6.  Set    infusion   on  water- 
bath,   keeping     temperature 
below  60°  C. 

7.  Add    peptone,    i%,    18 
gm. 


8.  After     ingredients     are 
dissolved,     titrate,    reaction 
probably +  2.3  to  +2.5. 

9.  Neutralize,      F  u  1 1  e  r's 
method. 


Ditto. 


Ditto. 


Ditto. 


Ditto. 


Ditto. 


Ditto. 


Ditto  and  sheet 
gelatin  10%,  180 
gm. 

Ditto. 

Probably +  4.0  to 
+  0.5. 

Ditto. 


Infuse  Jean  meat  20  hours 
with  its  own  weight  of  dis- 
tilled water  in  refrigerator. 

Say  1000  gm.  meat. 
"    1000  gm.  water. 


Ditto. 

i.e.  to  2000  gm. 


Ditto. 


Ditto. 

Say  reaction +  4. 2%. 


Ditto. 

Say  900  gm. 

Ditto. 


Add  peptone,  2%,  18  gm. 


Ditto. 

Prob. +4-5  to  +4.7. 


Ditto. 

To  the  900  gm.  of  meat 
infusion  (containing  now 
peptone  and  salt)  add  900 
gm.  of  the  3%  agar  jelly 
described  at  the  head  of 
this  column. 


10.  Heat  over  boiling  water  (or  steam)  bath  thirty  minutes. 

it.  Restore  weight  lost  by  evaporation  to  original  weight  of  filtered  meat 
infusion,  i.e.  that  on  which  the  percentage  of  peptone  and  salt,  etc.,  were  cal- 
culated, —  1800  gm.  in  each  case. 

12.  Titrate,  reaction  probably  +0.3  to  +0.5. 

13.  Adjust  reaction  to  final  point  desired,  +  1.5  per  cent. 

14.  Boil  five  minutes  over  free  flame,  with  stirring. 

15.  Add  water,  if  necessary,  to  make  up  loss  by  evaporation,  to  1800  gm. 

1 6.  Filter  through  absorbent  cotton,  passing  the  filtrate  through  the  filter 
repeatedly  until  clear. 

17.  Titrate  to  determine  whether  or  not  the  desired  reaction  has  been  main- 
tained. 

1 8.  Tube  and  sterilize. 


122  APPENDIX. 

N 
hydroxid  or  —  hydrochloric  acid.    As  a  rule,  the  reaction 

will  be  acid  and  the  alkali  may  be  used.  The  determina- 
tion should  be  made  not  less  than  three  times,  and  the 
average  of  the  results  taken. 


LACTOSE  AGAR. 

This  medium  is  made  in  the  same  manner  as  nutrient 
agar  except  that  2  per  cent  of  lactose  is  added  after  the 
final  filtration  and  the  reaction  is  adjusted  to  — .5. 

GLYCERINE  AGAR. 

For  this  medium,  5  per  cent  glycerine  is  added  to  nutri- 
ent agar  immediately  after  final  filtration. 

SUGAR  BROTH. 

The  most  important  and  widely  used  sugar  broth  is 
that  containing  dextrose,  although  lactose  and  saccharose 
and  occasionally  maltose  are  used  for  special  experi- 
mental work. 

Dextrose  broth  may  be  easily  prepared  by  adding 
2  per  cent  of  dextrose  to  ordinary  broth  just  before  the 
final  filtration,  and  making  the  reaction  neutral.  For 
the  preparation  of  lactose,  saccharose,  or  maltose  broth, 
however,  where  it  is  necessary  to  eliminate  dextrose,  the 
meat  infusion  after  filtration  is  placed  in  an  Erlerimeyer 
flask,  inoculated  with  an  active  broth  culture  of  B.  coli 


APPENDIX.  123 

and  incubated  for  twenty- four -hours  at  37°  C.,  whereby 
the  muscle  sugar  is  removed  by  fermentation. 

From  this  fermented  infusion,  broth  is  made  in  the 
ordinary  way,  2  per  cent  of  the  desired  sugar  added,  and 
the  medium  tubed  and  sterilized.  All  sugar  containing 
media  should  be  sterilized  with  great  care  to  avoid  break- 
ing down  the  sugar,  hence  it  is  advisable  to  use  the 
discontinuous  method,  heating  to  100°  C.  for  twenty 
minutes  on  three  successive  days,  rather  than  to  give  a 
single  heating  at  the  very  high  temperature.  There  is, 
however,  but  little  action  at  105°  C. 

It  is  the  custom  in  some  laboratories  to  prepare  broth 
and  sugar  solutions  separately,  and  mix  the  two  when  a 
sugar  test  is  to  be  made. 

MILK. 

Fresh  milk  in  a  tall  jar  is  placed  in  the  refrigerator  over 
night,  to  allow  the  cream  to  rise.  The  skim-milk  is  then 
siphoned  off  from  below  the  cream,  tubed,  and  sterilized 
at  110°  C.  for  fifteen  minutes,  or  on  three  successive  days 
at  1 00°  C.  for  twenty  minutes. 

LITMUS  MILK. 

This  is  prepared  as  above,  with  the  addition  of  \  c.c. 
blue  litmus  solution  to  give  a  faintly  alkaline  reaction. 

If  sterilized  at  a  high  temperature,  reduction  of  the 
litmus  may  take  place  with  loss  of  color,  but  oxidation 
will  follow  on  cooling  and  exposure  to  air. 


124  APPENDIX. 

POTATO. 

Large,  sound  potatoes  are  thoroughly  washed  and 
brushed  with  a  scrubbing-brush,  then  pared  and  cut  into 
cylinders  with  a  cork-borer.  After  paring,  the  potato 
should  be  kept  under  water  as  much  as  possible  to  pre- 
vent darkening.  The  cylinders  are  then  cut  diagonally  so 
that  each  produces  two  pieces  of  the  general  shape  of  a 
solidified  agar  or  serum  slant.  The  pieces  are  then  left 
in  cold,  running  water  for  several  hours,  after  which  they 
are  dropped,  broad  end  down,  into  test-tubes  containing 
a  small  piece  of  glass  rod  or  tubing  at  the  bottom  to  keep 
the  potato  out  of  the  watery  fluid  which  is  produced  by 
sterilizing.  Sterilize  for  twenty  minutes  at  100°  C.  on 
three  successive  days,  or  for  fifteen  minutes  at  110°  C. 

NITRATE   SOLUTION. 

A  stock  solution  is  prepared  by  dissolving  2  grams  C.  P. 
potassium  nitrate  in  100  c.c.  sterile  distilled  water.  This 
should  be  kept  in  the  ice-chest.  Five  c.c.  of  this  solution 
and  i  gram  peptone  are  added  to  i  liter  of  tap-water. 
The  solution  is  brought  to  a  boil,  filtered,  and  tubed. 

Care  must  be  taken  that  the  stock  solution  does  not 
become  reduced  to  nitrites  or  that  nitrite  is  not  present 
in  the  original  salt.  Sterilize  for  fifteen  minutes  at  120°. 

PEPTONE   SOLUTION. 

Ten  grams  peptone  and  5  grams  of  salt  are  dissolved  in 
i  litre  of  water;  the  solution  is  boiled,  filtered,  tubed,  and 
sterilized  for  fifteen  minutes  at  120°  C. 


125 


LOEFFLER'S  BLOOD  SERUM. 


This  medium  consists  of  3  parts  blood  serum  and 
i  part  of  i  per  cent  broth  with  reaction  +0.8.  The 
serum  is  obtained  from  fresh  beeves'  blood,  which  is  col- 
lected in  sterile  jars  and  allowed  to  stand  for  twenty-four 
hours  in  the  refrigerator  for  coagulation.  The  serum 
is  then  drawn  off,  filtered,  and  mixed  with  the  dextrose 
broth  in  the  proportion  above  indicated.  Hill  finds  that 
filtering  the  serum  through  the  coagulum  obtained  after 
adjusting  the  reaction  of  the  broth  gives  a  filtrate  which 
is  clear  and  almost  colorless  (Hill,  1899). 

Tubes  are  filled  with  the  mixture,  placed  in  trays  so 
that  the  desired  slant  is  obtained,  and  carefully  heated 
in  a  Koch  coagulator  containing  cold  water  in  the  water- 
jacket.  This  water  is  brought  to  a  boil  and  kept  boiling 
for  three  hours.  Repeating  this  process  on  three  suc- 
cessive days  solidifies  the  serum  so  that  it  may  be  subse- 
quently sterilized  in  flowing  steam  for  twenty  minutes 
on  three  successive  days. 

PHENOL  BROTH. 

To  1000  c.c.  water  add  separately,  and  in  the  following 
order,  100  grams  dextrose,  50  grams  peptone,  2.5  grams 
phenol.  Heat  until  all  constituents  are  dissolved,  boil 
for  fifteen  minutes,  and  sterilize  for  fifteen  minutes  at 
110-120°  C. 


126  APPENDIX. 


NEUTRAL  RED  BROTH. 

To  neutral  broth  is  added  0.5  per  cent  dextrose  and 
i  per  cent  of  a  0.5  per  cent  aqueous  solution  of  Griibler's 
neutral  red.  Sterilize  at  100°. 

MACCONKEY'S  MEDIA. 

A.  Agar. 

Agar 1.5  grams 

Sodium  taurocholate  (pure) 0.5  gram 

Peptone 2.0  grams 

Water 100.0  c.c. 

This  is  boiled,  clarified,  and  filtered  as  usual,  then 
i.o  gram  lactose  is  added,  and  the  medium  tubed  and 
sterilized  for  three  successive  days  at  100°. 

B.  Broth. 

Sodium  taurocholate  (pure) 0.5  gram 

Peptone 2.0  grams 

Glucose 0.5  gram 

Water 100.0  grams 

Boil,  filter,  and  add  sufficient  neutral  litmus,  fill  fer- 
mentation-tubes, and  sterilize  at  100°. 

Litmus  Solution. — To  one-half  pound  of  litmus  cubes 
add  enough  water  to  more  than  cover,  boil,  and  decant 
off  the  solution.  Repeat  this  operation  with  successive 
small  quantities  of  water  until  from  3  to  4  liters  of  water 
have  been  used  and  the  cubes  are  well  exhausted  of  color- 
ing matter.  Pour  the  decantations  together  and  allow 
them  to  settle  overnight.  Siphon  off  the  clear  solution. 
Concentrate  to  about  i  liter  and  make  the  solution  de- 
cidedly acid  with  glacial  acetic  acid.  Boil  down  to  about 


APPENDIX.  127 

J  liter  and  make  exactly  neutral  with  caustic  soda  or 
potash.  To  test  for  the  neutral  point,  place  one  drop  of 

N 

the  solution  in  a  test-tube.     One  drop  of  —  HC1  should 

20 

N 
turn  the  drop  red,  while  one  drop  of  —  NaOH  should  turn 

it  blue.  Filter  the  solution  and  sterilize  at  110°  C.  This 
solution  should  be  added  to  the  media  just  before  use  in 
the  proportion  of  about  J  c.c.  to  5  c.c.  of  medium. 


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AUTHOR  INDEX. 


PAGE 

Abba 78 

Abbott 25 

Amyot 75 

Andrewes 5  *»  54,  100 

c*~ 

Baker 102 

Bassett 75 

Beckmann 76 

Belcher 107 

Belitzer 75 

Blachstein 78 

Blunt 13 

Bolton 37 

Brotzu 75 

Brown 83 

Bruns 78 

Buchner 13 

Burri 82 

Cambier 25,  31 

Chick 45,  fo.  85 

Clark 40,  87 

Copeland 60 

Cramer 38 

Dombrowsky 53 

Downes 13 

Duclaux 7 

Dunbar 55 

143 


144  AUTHOR  INDEX. 

PAGE 

Dunham  ..............................................    .  ,   40,  55 


Egger  .......................................  ,  ..............      15 

Ellms  ......................................................     25 

Eisner  ..........................................    ..........     50 

Escherich  .................................................  58,  74 

Fischer  ...................................................  15,  53 

Flatau  .....................................................      53 

Frankland  .......................................  9,  10,  25,  54,  56 

Fremlin  ................  ....................................     74 

Freudenreich  ..............................................  12,  82 

Frost  ......................................................     70 

Fuller  ..................................  20,  29,  40,  73,  85,  119,  120 

Gage  ...................  19,  21,  30,  61,  63,  66,  72,  73,  87,  95,  98,  101 

Garre  .....................................................      12 

Gartner  ..................................................  23,  37 

Gotschlich  ................................  ..................     56 

Hammerl  .........................................  ..........      83 

Hankin  ....................................................      50 

Hansen  ....................................................    113 

Heider  ....................  .............................  116,  117 

Heraeus  .  .  .  .................................................     23 

Hesse  .....................................................      19 

Hill  .............................................  25,  98,  120,  125 

Hoag  ......................................................     75 

Horrocks  ..................  .  ..............  .....  12,  15,  85,  101,  105 

Houston  ............................  80,  84,  loo,  105,  107,  113,  115 

Hunnewell  ........................................  64,  91,  96,  101 

Irons  ...............................................  61,  63,  95,  98 

Janowski  ...................................................       8 

Johnson  ...................................    ................      73 

Jordan  ...............................  II,  13,  14,  27,  39,  88,  89,  117 

Keith  ......................................................     74 

Klein  ........................................  50,  80,  106,  107,  113 

Koch  ......................................................     55 


AUTHOR  INDEX.  US 

PAGZ 

Kolle 56 

Kruse 76 

Kubler.    53 

Kusel 73 

Laws 51,  54,  100 

Le  Gros 101 

Levy 78 

Loeffler ...    55.1-5 

MacConkey 98,  126 

Makgill 97 

Maschek 1 5,  23 

Mathews 46,  59 

Miquel 8,  9,  25,  31,  35,  39,  82 

Moore 75 

Moroni 78 

Neufeld 53 

Nibecker 10,  46,  66,  97,  101 

Niedner 19 

Nuttall 106 

Osgood 1 16 

Pakes 84 

Papasotiriu 80 

Parietti 49 

Pennington 73 

Pctruschky 92 

Phelps 19-  21,  30,  66,  72,  98 

Poujol 77 

Prausnitz. 39 

Prescott 16,  17,  29,  61,  80,  101,  102 

Procaccini 13 

Pusch 92 

Rapp 13,  14 

Refik 76 

Reinsch 29 

Kemlinger 54 


146  AUTHOR  INDEX. 

PAGE 

Reynolds 62 

Rideal 46 

Riedel 26 

Rothberger 97 

Russell 75 

Savage 97 

Scheurlen 1 1 

Schneider 54 

Schottelius 55 

Sedgwick 5,  12,  16,  17,  29,  57,  59 

Shuttleworth 41 

Smith 60,  74,  82,  94 

Sternberg 36 

Stokes 98 

Thomson 50 

Tiemann 23,  37 

Tissandier 7 

Wathelet 51 

Weissenfeld 78 

Welch 106 

Whipple 9,  20,  26,  30,  31,  58,  65,  68,  96,  113,  115 

Widal 53 

Winslow 10,  12,  22,  41,  46,  64,  66,  73,  91,  96,  97,  101,  107,  113 

Wollfhugel 15,  26 

Woodman 113 

Wright 75 

Wurtz 44,  59 

Zagari 12 


SUBJECT  INDEX. 


PAOB 

Absorption  of  CO,  70 

Acid  formers  in  Boston  sewage 45 

production  by  B.  subtilis 45 

Acidity  of  media 29     ^ 

Agar  media 30 

plate  count 44     r+* 

preparation  of 120,  121     £Q 

streak,  types  of  growth 67    ' 

Agglutination 53 

Air,  dust  in 7    2S 

American  Public  Health  Association,  standard  procedure. . .  19,  20,  28,    j 

30,  31,   II9,   120     ^ 

Amines,  formation  of  by  B.  coli 66  *£ 

Ammonia,  formation  of  by  B.  coli 66  0« 

Ammonium  compounds,  decomposition  of 5  * 

Antagonism 12 

Bacillus  aerogenes 108  ^ 

anthracis,  isolation  from  water 56  *" 

cloacae 91,  92,  108 

coli,  as  a  saprophyte. 76 

,  as  direct  evidence  of  sewage 58 

,  atypical  forms 68 

,  biochemical  characters 51,  58,  59 

,  characteristics  of. 67 

,  colonies  on  agar 67 

,  comparison  of  methods  for  isolation 61,  63 

,  decomposition  of  sugars 44 

,  detection  ofj  in  presence  of  streptococci 104 


148  SV EJECT  INDEX. 

PAGE 

Bacillus  coli,  determined  by  animal  inoculation 78 

,  distribution  of 81 

,  distribution  of,  in  unpolluted  waters 76-79 

,  distribution  of,  in  various  animals 74 

,  distribution  in  various  waters 85 

,  effect  of  dilution  on  growth  in  litmus  lactose  agar. ...     66 

,  from  cereals 82 

,  gas  ratio 95 

,  general  distribution  of,  in  nature 76 

,  growth  on  agar  streak 67 

in  effluents  of  Lawrence  filter 87 

in  filter  effluents 86 

in  large  samples 82 

in  Merrimac  River 86 

in  mud 45 

in  normal  intestine 58 

in  polluted  waters 89,  91 

in  refuse  from  tanneries,  grist-mills,  and  dairies 82 

in  river  water 83,  92 

in  Severn  River 45 

in  sewage 89,  92 

in  soil 84 

in  spring  water 78,  88 

in  stored  sewage 101 

,  in  unpolluted  waters 78,  91,  97 

in  well  waters  78,  92 

,  isolation  of,  by  Escherich 58 

,  isolation  of  pure  cultures 65 

,  isolation  of,  by  litmus  lactose  agar 59 

,  morphology  of. 58 

,  on  cereals 80 

,  on  hands 22 

,  overgrowth  by  streptococci 102,  103,  104 

,  overgrowth  during  preliminary  incubation  .   ...  62,  64,  65 

,  pathogenesis  for  guinea-pigs , 59 

,  pathogenicity  of 78 

,  percentage  of  cultures  giving  positive  tests  in  sub- 
culture      72 

,  positive  isolations  by  different  methods 63 

,  presumptive  tests  for 94 


SUBJECT  INDEX.  149 

PAGE 

Bacillus  coli,  presumptive  tests  in  various  amounts  of  water 96 

,  qualita*-'  ve  analysis  for 69,  70 

,  quantitative  estimation  of 35,  82 

,  relation  to  B.  acidi  lactici 80 

,  significance  of 1 12 

,  significance  of,  in  samples  of  different  size 87 

,  significance  of,  in  water 74 

,  standards  for  drinking  water 85 

,  standards  for  presumptive  test 97 

,  sub-cultures  of,  in  qualitative  analysis 70 

,  susceptibility  to  phenol 59 

,  time  of  gas-production  in  dextrose  broth  by 61 

,  variations  in  cultural  reactions 73 

,  varieties  of 68 

Bacillus  dysenterise 53 

enteritidis  sporogenes 106 

mycoides 66 

,  growth  on  agar  streak 67 

sporogenes,  as  index  of  pollution 106 

,  cultural  characteristics 106,  107 

in  sewage 107 

,  method  for  detection  of,  in  water . .  106 

,  significance  of 107 

typhi,  biochemical  characters  of. 51 

,  distribution  in  the  environment 54 

in  ice 12 

,  isolation  of. 49 

,  life  of,  in  water 54 

Bacteria,  classes  of,  in  relation  to  nutrition 37 

,  comparative  numbers  of,  growing  at  20°  and  37° 47 

,  definition  of  species 67 

,  developing  on  various  media 30 

,  distribution  of     .    3 

,  distribution  of,  in  sea-water 10 

,  distribution  in  water ~«6 

,  diurnal  variation 14 

fermenting  lactose,  in  normal  waters 46,  48 

,  in  polluted  waters 45,  48 

growing  at  body  temperature 43 

in  Boston  tap- water 9,  38 


SUBJECT  INDEX. 

PAGE 

Bacteria  in  Boston  sewage 45 

in  Cambridge  supply 10 

in  Chicago  drainage  canal 39 

in  Connecticut  River 42 

in  deep  wells 17,  40 

in  driven  wells 17,  40 

in  filter  effluents 40 

in  Framingham  supply 42 

in  ground  water 15,  39 

in  ground  waters,  peculiar  character  of 18,  39 

in  Hartford  supply 42 

in  ice 12 

in  Isar  River 39 

in  Lake  Champlain 10 

in  Lake  of  Lucerne  .   10 

in  Lake  Zurich 38 

in  lakes  and  ponds 10 

in  Loch  Katrine 10 

in  Loch  of  Lintralthen 10 

in  Lynn  supply 10 

in  Medford  supply 10 

in  Merrimac  River 9 

in  Newport  supply 42 

in  Ohio  River 41 

in  Ourcq  River 9 

in  Peabody  supply 10 

in  Plymouth  supply 10 

in  polluted  streams 39 

in  polluted  waters,  changes  during  storage 27 

in  polluted  well 42 

in  rain 8 

in  relation  to  food  supply 14 

in  Salem  supply 10 

in  sea-water  at  Naples 10 

in  sea-water  at  Wood's  Hole 10 

in  Seine 39 

in  shallow  wells 15,  16 

in  snow 8 

in  springs 15,  16 

in  stored  samples,  multiplication  of 25~27 


SUBJECT  INDEX.  151 

PAGE 

Bacteria  in  surface  wash 8 

in  surface  waters 9 

in  Taunton  supply 10 

in  Thames  River 9 

in  Wakefield  supply    10 

in  water,  relation  to  organic  matter 37,  38,  in 

,  significance  of  number  of 38 

in  well  water,  effect  of  pumping  on 23 

,  intestinal 99 

f  liquefying 108 

,  metabolism  of 4 

,  multiplication  of,  in  spring  water 16 

,  peculiar  groups  in  ground  water 18,  39 

,  reduction  of,  in  streams 8 

,  relation  to  environment.^ 3,  14 

organic  matter  in  water 6 

waste  materials 4 

,  seasonal  distribution  in  surface  waters 9 

which  do  not  grow  on  ordinary  media 19 

Bacterial  standards  for  filter  plants 41 

Bacteriological  analyses,  value  of 41 

analysis  of  water,  certainty  of 42,  115 

,  comparison  with  chemical 113 

,  delicacy  of 113 

,  directness  of 1 13 

,  distribution  of  sewage  in  water. . .  116 

ground  waters 113,  114,  115 

examination  of  water,  applicability  of. 116 

Berkefeld  filter  to  concentrate  organisms 50 

Biochemical  characters  of  B.  coli 51 

Blood  serum,  preparation  of. .    125 

Body  temperature,  count,  Boston  sewage 45 

,  Charles  River 45 

of  various  samples 47 

,  polluted  water 45 

,  significance  of 112 

organisms  growing  at 43 

Boston,  B.  spor6genes  in  sewage 107 

,  bacteria  in  deep  wells 17 

r  course  of  sewage  in  harbor. 116 


152  SUBJECT  INDEX. 

PAGE 

Boston  sewage,  bacteria  in 45 

tap-water,  bacteria  in 9,  38 

Brookline  tap-water,  bacteria  in 47 

Brooks,  bacteria  in 47 

Cambridge  tap-water,  bacteria  in 10,  47 

Carbon  dioxide,  absorption  of 70 

Cats,  B.  coli  in 74,  75 

Cereals,  B.  coli  in .  80 

Character  of  colonies,  importance  of,  in  ground  waters 40 

Charles  River  above  Boston,  body  temperature,  count 45 

Chemical  analysis  of  water,  comparison  with  bacteriological 113 

Chemical  examination  of  water no 

,  applicability  of 1 16 

Chicago  drainage  canal,  B.  coli  in 89 

,  bacteria  in 39 

,  routine  bacteriological  analyses 41 

Chichester,  well  waters  in 115 

Cleaning  mixtures 22 

Cold,  action  of,  upon  bacteria , 12 

Colon  bacilli  in  Manchester  Ship  Canal 45 

,  overgrown  by  streptococci 92 

Colon  bacillus,  as  direct  evidence  of  sewage 58 

,  biochemical  characters 58,  59 

,  gas  ratio 58 

,  importance  of  number  of 82 

,  in  normal  intestine 58 

,  isolation  of,  by  litmus  lactose  agar 59 

,  morphology  of 58 

,  pathogenesis  in  guinea-pigs 59 

,  preliminary  enrichment  for 60 

,  quantitative  estimation  of. 60 

,  susceptibility  to  phenol 59 

Comparability  of  quantitative  results 20 

Composition  of  media,  effect  on  bacterial  counts 30 

Connecticut  River,  bacteria  in 42 

Constantinople,  B.  coli  in  water  supplies 76 

Counting 32,  33 

Cows,  B.  coli  in 74>  75 

Cycle  of  organic  nitrogen 5 


SUBJECT  INDEX.  153 

PAGE 

Daily  analyses,  at  Chicago,  etc 41 

Danube  River,  distribution  of  sewage  in 116 

Dedham  tap-water,  bacteria  in 47 

Deep  wells 15 

,  bacteria  in 17 

Depths  below  surface,  taking  samples  from 24 

Desplaines  River,  B.  coli  in 89 

Dextrose  broth,  for  enrichment  of  B.  coli 61 

,  method  for  isolation  of  B.  coli 63 

,  preparation  of. 122 

Dilution  of  samples  in  plating 28 

Distribution  of  bacteria  in  sea-water II 

water 6 

Diurnal  variation  in  bacteria 14 

Dogs,  B.  coli  in 74,  75 

Driven  wells,  bacteria  in 40,  47 

Dust  in  air 7 

Egypt,  cholera  in 56 

Elbe  River,  cholera  bacilli  in 55 

Eisner's  medium 54 

Enrichment  culture,  for  B.  typhi 49 

processes,  disadvantage  of. 50 

process,  effect  upon  B.  coli 50 

Environment,  effect  of 14 

Esmarch  process 34 

Examination  of  spring  waters  for  B.  coli 88 

Feces,  streptococci  in 101 

Fermentation,  characteristic  gas  formula  of  B.  coli 75 

,  gas  formula  of  B.  coli 95 

of  sugars  by  B.  subtilis 45 

,  variations  in 73 

Field,  plating  in 34 

Filter  effluent,  B.  coli  in 86,  87 

Filtration  of  water 40 

Fishes,  B.  coli  in 75,  76 

Food  supply,  effect  of,  upon  bacteria II,  14 

Fowls,  B.  coli  in 75 

Fox  River,  B.  coli  in 90 


154  SUBJECT  INDEX. 

PAGE 

Framingham  supply,  bacteria  in 42 

Freezing  of  bacteria 12 

Gas  formula 95 

Gas  measurement,  in  fermentation  tube 70 

Gasometer 70 

Gas  production  in  dextrose  broth  by  B.  coli,  time  of 61 

Gelatin 119,  121 

,  liquefaction  of 71 

media 30 

,  physical  condition  of. 30 

Glycerin,  agar,  preparation  of. 122 

Glycerin  media 30 

Goats,  B.  coli  in 74,  75 

Ground  waters 15 

,  bacteria  in 39 

,  bacteriological  analysis  of 1 13,  114,  115 

,  character  of  bacteria  in 18,  39 

Hamburg,  cholera  epidemic 55 

Hartford  supply,  bacteria  in 42 

Hogs,  B.  coli  in 74,  75 

Horses,  B.  coli  in 74,  75 

Hudson  River,  B.  coli  in 83 

Ice,  bacteria  in 12 

Illinois  River n 

,  B.  coli  in 90 

,  distribution  of  sewage  in 117 

Incubation,  conditions  of 31 

of  agar  plates  at  body  temperature 44 

,  period  of 31,  32 

Indol  reaction 55 

test 71 

Interpretation  of  analyses 117 

Interval  between  sampling  and  examination 28 

Intestinal  bacteria 99 

Isar.  River,  bacteria  in 13,  14,  39 

Isolation  of  B.  coli,  Chicago  methods 89 

,  definition  of  species. 67 


SUBJECT  INDEX.  155 

PAGE 

Isolation  of  B.  coli,  examination  of  subcultures 71 

,  from  large  samples 63,  64,  65 

,  growth  on  agar  streak 67 

,  Mass.  Inst.  Tech.  record  blank 69 

,  Mass.  State  Board  of  Health  methods 69 

,  necessity  of  standard  methods 69 

,  percentage  of  cultures  passing  various  tests 72 

,  period  of  preliminary  enrichment 62 

,  phenol  broth  method 62 

,  preliminary  enrichment 61 

,  separation  of  pure  cultures 65 

,  significance  of  large  samples 87,  91 

,  significance  of  quantitative  results 81 

,  variations  in  cultural  reactions 73 

streptococci - 102 

Jewell  filter,  bacterial  efficiency 41 

Kiel,  bacteria  in  wells  at 15,  16 

Lactic  acid  bacteria 79 

Lactose-agar  plate  as  a  presumptive  test 97 

plates 44 

,  use  in  isolation  of  B.  coli 60,  65 

,  preparation  of 122 

broth,  preparation  of. 122 

,  fermentation  of,  by  B.  coli 58,  59 

-fermenting  bacteria  in  polluted  waters 48 

-fermenting  organisms  in  normal  water 48 

Lake  Champlain,  bacteria  in 10 

of  Lucerne,  bacteria  in IO 

Zurich,  bacteria  in 38 

Lakes,  bacteria  in IO 

Lawrence  city  filter,  bacterial  efficiency  of 40,  87 

Leitmeritz,  bacteria  in  wells  at 15 

Light,  effect  of,  upon  bacteria 1 1,  13 

in  streams 13 

Liquefying  bacteria 108 

,  phenolated,  for  B.  coli  isolation 60 

Litmus  lactose  agar  plate,  incubation  of 66 

plate,  isolation  of  B.  coli  by 59 


156  SUBJECT  INDEX. 

PAGE 

Litmus  lactose  agar  plate,  technique  of 45 

,  red  colonies  in 66 

Litmus  milk,  preparation  of 123 

Liverpool  tap-water,  B.  coli  in 85 

Loch  Katrine,  bacteria  in 10 

Loch  of  Lintralthen,  bacteria  in 10 

Loeffler's  blood  serum 125 

Lynn  tap-water,  bacteria  in 10,  47 

MacConkey's  media,  preparation  of 126 

Mainz,  bacteria  in  wells  at 15 

Maltose  broth,  preparation  of. 122 

Manchester  ship  canal,  B.  coli  in 45,  85 

Massachusetts  Institute  of  Technology,  blank  for  recording  bacterio- 
logical analysis 69 

Massachusetts  State  Board  of  Health 9,  72,  86,  88 

,  B.  coli  isolation 87 

,  bacteriological  counts 40 

,  examination  of  springs 16 

,  methods  for  isolation  of  B.  coli     69 

Mechanical  filtration 40 

Medford  tap- water,  bacteria  in 10,  47 

Media,  alkaline,  for  isolation  of  Sp.  cholerae 55 

,  bacteria  developing  on  various 30 

,  comparability 20 

,  composition  of 29 

,  limitations  of  ordinary 19 

,  percentage  of  bacteria  developing  on  various. 21 

,  practical  requirements  for 20 

,  reaction  of 29 

,  standard  methods 119,  121 

Merrimac  River,  bacteria  in .  9,  40 

Metabolism  of  different  classes  of  bacteria 37 

Mice,  B.  coli  in 74 

Micro-organisms,  effect  of,  upon  bacteria 1 1,  12 

Milk,  preparation  of 123 

test 71 

Milton  tap-water  bacteria  in 47 

Mississippi  River,  B.  coli  in 83,  90 

Missouri  River,  B.  coli  in 90 


SUBJECT  INDEX.  157 

PAGE 

Mohawk  River,  B.  coli  in 83 

Moisture  in  incubators 31 

Montsouris,  bacteria  in  air 8 

Mud,  colon  bacilli  in 45,  85 

Multiplication  of  bacteria  in  spring  waters 16,  25 

in  stored  samples 25~27 

Mur  River,  B.  coli  in 83 

Nahrstoff  Heyden  agar 19,  20,  21,  30 

Naples,  bacteria  in  sea-water  at 10 

Neumark,  typhoid  bacillus  in  well  at 53 

Neutral-red 97 

broth,  preparation  of 126 

Newburyport  tap-water,  bacteria  in 47 

Newport  supply,  bacteria  in 42 

New  York  Board  of  Health 82 

,  examination  of  rivers  for  B.  coli 83 

Nitrate  solution,  preparation  of 124 

test 71 

,  variations  in 73 

Nitrification 5 

Nitrifying  organisms 5 

Nitrites 5 

Nitrogen,  cycle  of  organic 5 

Normal  waters,  lactose  fermenting  bacteria  in 48 

Ohio  River,  B.  coli  in 85 

,  bacteria  in 41 

Organic  matter,  decomposition  of 4,  in 

in  water,  relation  of  bacteria  to 37 

,  oxidation  of 5 

,  relation  of  bacteria  to 6 

Osmotic  pressure,  effect  of  upon  bacteria 1 1 

Ourcq  River,  bacteria  in 9 

Overgrowth 62,  64 

Oxidation  of  organic  matter 5 

Oxygen,  effect  of,  upon  bacterial  counts 31 

Para-colon  organisms 78,  91,  92 

Para-typhoid  organisms 78 

Parietti's  solution 50 


158  SUBJECT  INDEX. 

PAGE 

Paris,  bacteria  in  air  of. 8 

Parma,  B.  coli  in  water  supply  of. 78 

in  wells  and  springs  near 78 

Peabody  tap-water,  bacteria  in IO,  47 

Peptone  method  for  isolation  of  cholera  bacillus 56 

solution,  preparation  of 124 

test 71 

Period  of  incubation 31 

Phenol  broth,  composition  of 62 

,  method  for  isolation  of  B.  coli 62 

,  preparation  of. 125 

Phenolated  gelatin,  use  of,  for  typhoid  isolation 50 

lactose  litmus  agar 60 

Plating 28 

in  the  field „ 34 

Plymouth  tap-water,  bacteria  in. 10,  47 

Polluted  waters,  bacterial  changes  during  storage 27 

,  blood  temperature  count 45 

,  lactose  fermenting  bacteria  in 48 

,  specific  pathogenes  in 49 

Ponds,  bacteria  in 10,  47 

Pools,  bacteria  in , 47 

Potato,  preparation  of 124 

gelatin,  use  for  typhoid  bacteria 50 

Presumptive  tests  for  B.  coli 94  et  seq. 

Proteus  organisms 108 

Protozoa 1 2 

Prussia,  regulations  regarding  filter  plants 41 

Pseudocolon  bacilli 68 

Pump,  sampling  from 23 

Pumping,  effect  of,  upon  bacterial  content  of  well  water 23 

Quantitative  analysis   arbitrary  standards  for 35 

composition  of  media 29 

conditions  of  incubation 31 

counting 33 

dilution 28 

general  procedure 21 

interpretation  of 35 

media  for 19 


SUBJECT  INDEX.  1 59 

PAGE 

Quantitative  analysis,  period  of  incubation 31 

,  plating 28-29 

,  procedure  for 21 

,  recording  results 33 

,  sampling 21-24 

,  storage  of  samples 25 

bacteriological  analysis,  significance  of Ill 

Rabbits,  B.  coli  in 74,  75 

Rain,  bacteria  in 8,  47 

,  contamination  of  surface  water  by 9 

Reaction  of  media 29 

Recording  quantitative  results 33 

Red  colonies  in  litmus  lactose  agar 66 

Relation  of  B.  coli  to  B.  acidi  lactici 80 

Rellingen,  typhoid  bacillus  in  well  at 53 

Saccharose  broth,  preparation  of. 122 

Salem  tap-water,  bacteria  in 10,  47 

Sample  bottles,  cleaning  of 22 

,  handling 22 

,  size  of,  effect  of,  upon  multiplication  of  bacteria. ...     27 

,  sterilization  of 22 

Sampling,  for  bacteriological  analysis 21-25 

,  necessity  for  care  in 37 

Sand  nitration 40 

Sangamon  River,  B.  coli  in 90 

Sanitary  inspection 109 

Research  Laboratory,  bacteria  in  Boston  sewage 45 

Sea-water,  bacteria  in 10 

Seasonal  distribution  of  bacteria  in  surface  waters 9 

variation  of  bacterial  content  in  surface  waters 9,  38 

Sedimentation  of  bacteria n 

Seine,  bacteria  in 39 

Self-purification  of  lakes  and  ponds 10 

of  streams 8,  13,  39,  113 

Severn  River,  colon  bacilli  in 45,  85 

Sewage,  B.  coli  in 89 

,  bacteria  in 45 

,  distribution  of,  in  water 116,  117 

pollution,  bacteria  in  relation  to 39 


160  SUBJECT  INDEX. 

PAGE 

Sewage,  streptococci  in 62,  100,  101 

,  typhoid  bacillus  in 51 

Shallow  wells,  bacteria  in 15,  16 

Sheep,  B.  coli  in 75 

Shiga  bacillus 53 

Significance  of  B.  coli 89 

in  water 76  et  seq.,  93 

,  necessity  for  quantitative  results. ...     81 

of  blood  temperature  count 43 

of  quantitative  analysis 38 

of  streptococci  in  water 105 

Snow,  bacteria  in 8,  47 

Sodium  taurocholate  medium 98 

Specific  pathogenes  in  polluted  water 49 

Spirillum  cholerae  in  Elbe  River 55 

,  isolation  of 54,  55 

of  Asiatic  cholera,  isolation  from  polluted  water 49 

Spore-forming  pathogenes  in  water,  isolation  of. 56 

Spring  waters,  multiplication  of  bacteria  in , . . . .     16 

Springs,  bacteria  in 15,  16,  47 

Standard  for  interpreting  presumptive  test 97 

Sterilization  of  gelatine,  effect  of 30 

sample  bottles 22 

Storage n 

,  effect  on  sample 37 

,  multiplication  of  bacteria  during 25-27 

of  polluted  waters 27 

of  samples,  allowable  maximum 28 

Strassburg,  B.  coli  in  water  of 76 

Streams,  bacteria  in 39 

,  sedimentation  of  bacteria  in 1 1 

Streams,  self-purification  of. 8,  13 

Streptococci  as  indicative  of  pollution 100 

,  colonies  on  litmus  lactose  agar 66 

,  cultural  characters 99 

,  decomposition  of  sugars  by 44 

,  detection  of,  in  presence  of  B.  coli 104 

(  growth  on  agar  streak 67 

,  habitat  of 101 


SUBJECT  INDEX.  161 

PAGE 

Streptococci  in  feces 101 

in  polluted  waters 92,  102 

in  sewage 101 

in  sewage  polluted  rivers 100 

in  stored  sewage 101 

in  unpolluted  water 91,  101 

,  isolation  of. 102 

,  overgrowth  of  B.  coli  by 102,  104 

,  relation  to  B.  coli 102,  103,  104 

sewage  pollution 105 

,  sewage 62,  100 

,  significance  of 1 12 

,  types  of  colonies  on  litmus  lactose  agar 66 

Streptococcus  erysipelatos 99 

Sudbury  River,  chemical  and  bacteriological  examination 113 

Sugar  broth,  preparation  of 122 

Sugars,  decomposition  of. 44 

Surface  wash,  bacteria  in 8 

water,  sampling  of. 24 

,  bacteria  in 9,  39,  47 

Tap-water  sampling 22 

Taunton  tap-water,  bacteria  in 10,  47 

Temperature,  effect  of,  upon  bacteria II,  12 

,  effect  upon  multiplication  of  bacteria  in  stored  samples, 

26,27 

of  incubation 31 

Thames  River,  bacteria  in 9 

Titration  of  media .    120 

Toronto  supply,  bacteria  in 41 

Toxic  products 12 

Turin,  B.  coli  in  unpolluted  waters  near 78 

Turkeys,  B.  coli  in 75 

Typhoid  bacillus  in  ice 12 

in  sewage 57 

in  well  at  Neumark 53 

Rellingen    54 

isolation  from  polluted  water 49 

,  reported  isolation  from  water 53 

fever,  spring  epidemics 38 


162  SUBJECT  INDEX. 

PAGE 

Urea,  decomposition  of 5 

Value  of  isolated  analyses 41 

Variation  of  B.  coli 68 

Variations  in  cultural  reactions 73 

Wakefield  and  Stoneham  tap-water,  bacteria  in 10,  47 

Water,  B.  coli  in  different  grades  of 96 

in  polluted  and  unpolluted 63 

bacteria,  peculiar  metabolism  of 37 

,  bacteriological  analysis  of 69 

,  chemical  composition,  relation  of  bacteria  to 37,  38 

,  chemical  examination  of 1 10 

,  complete  bacteriological  analysis 112 

,  distribution  of  bacteria  in „ 6 

purification 40 

supplies,  sanitary  inspection  of 109 

Waters,  bacteria  in  various  classes  of. 21 

,  classification  of 6 

Well,  typhoid  bacillus  in,  at  Neumark 53 

at  Rellingen 53 

water,  bacteria  in 42 

Wells,  bacteria  in 47 

Westerly  tap-water,  bacteria  in 47 

Widal  reaction 53 

Wood's  Hole,  bacteria  in  sea-water  at 10 

Wurtz  litmus  lactose  agar 59 


SHORT-TIT.LE      CATALOGUE 

OF   THE 

PUBLICATIONS 


OP 


JOHN    WILEY    &    SONS, 

NEW  YORK. 
LONDON:   CHAPMAN  &  HALL,  LIMITED. 


ARRANGED  UNDER  SUBJECTS. 


Descriptive  circulars  sent  on  application.  Books  marked  with  an  asterisk  are 
sold  at  net  prices  only,  a  double  asterisk  (**)  books  sold  under  the  rules  of  the 
American  Publishers  Association  at  net  prices  subject  to  an  extra  charge  for 
postage.  All  books  are  bound  in  cloth  unless  otherwise  stated. 


AGRICULTURE. 

Armsby's  Manual  of  Cattle-feeding i2mo,  Si  75 

Principles  of  Animal  Nutrition 8vo,  4  oo 

Budd  and  Hansen's  American  Horticultural  Manual: 

Part  I. — Propagation,  Culture,  and  Improvement i2mo,  i  50 

Part  II. — Systematic  Pomology lamo,  i  50 

Downing's  Fruits  and  Fruit-trees  of  America 8vo,  5  oo 

Elliott's  Engineering  for  Land  Drainage I2mo,  i  50 

Practical  Farm  Drainage I2mo,  i  oo 

Green's  Principles  of  American  Forestry i2mo,  i  50 

Grotenfelt's  Principles  of  Modern  Dairy  Practice.     (Woll.) I2mo,  2  co 

Kemp's  Landscape  Gardening i2mo,  2  50 

Maynard's  Landscape  Gardening  as  Applied  to  Home  Decoration i2mo,  i  50 

Sanderson's  Insects  Injurious  to  Staple  Crops i2mo,  i  50 

Insects  Injurious  to  Garden  Crops.     (In  preparation.) 

Insects  Injuring  Fruits.     (In  preparation.) 

Stockbridge's  Rocks  and  Soils 8vo,  2  50 

Woll's  Handbook  for  Farmers  and  Dairymen i bmo,  i  50 

ARCHITECTURE. 

Baldwin's  Steam  Heating  for  Buildings i2mo,  2  50 

Berg's  Buildings  and  Structures  of  American  Railroads 4to,  5  oo 

Birkmire's  Planning  and  Construction  of  American  Theatres 8vo,  3  oo 

Architectural  Iron  and  Steel 8vo,  3  50 

Compound  Riveted  Girders  as  Applied  in  Buildings 8vo,  2  oo 

Planning  and  Construction  of  High  Office  Buildings • 8vo,  3  50 

Skeleton  Construction  in  Buildings 8vo,  3  oo 

Briggs's  Modern  American  School  Buildings 8vo,  4  oo 

Carpenter's  Heating  and  Ventilating  of  Buildings 8vo,  4  oo 

Freitag's  Architectural  Engineering.     2d  Edition,  Rewritten 8vo,  3  50 

Fireproofing  of  Steel  Buildings 8vo,  2  50 

French  and  Ives's  Stereotomy 8vo,  2  50 

Gerhard's  Guide  to  Sanitary  House-inspection i6mo,  i  oo 

Theatre  Fires  and  Panics i2mo  i  50 

1 


Holly's  Carpenters'  and  Joiners'  Handle;. A i8mo,  o^  75 

Johnson's  Statics  by  Algebraic  and  Graphic  Methods 8vo  2  oo 

Kidder's  Architect's  and  Builder's  Pocket-book.     (Rewritten  edition  in  preparation.) 

Merrill's  Stones  for  Building  and  Decoration 8vo,  5  oo 

Monckton's  Stair-building 4to,  4  oo 

Pattern 's  Practical  Treatise  on  Foundations 8vo,  5  oo 

Siebert  and  Biggin's  Modern  Stone-cutting  and  Masonry 8vo,  i  50 

Snow's  Principal  Species  of  Wood 8vo,  3  50 

Sondericker's  Graphic  Statics  with  Applications  to  Trusses,  Beams,  and  Arches. 

8vo,  2  oo 

Wait's  Engineering'and  Architectural  Jurisprudence 8vo,  6  oo 

Sheep,  6  50 

Law  of  Operations  Preliminary  to  Construction  in  Engineering  and  Archi- 
tecture   8vo,  5  oo 

Sheep,  5  50 

Law  of  Contracts 8vo,  3  oo 

Woodbury's  Fire  Protection  of  Mills 8vo,  2  50 

Worcester  and  Atkinson's  Small  Hospitals,  Establishment  and  Maintenance, 
Suggestions  for  Hospital  Architecture,  with  Plans  for  a  Small  Hospital. 

i2mo,  i  25 

The  World's^Oolumbian  Exposition  0^1893 Large  4to,  i  oo 


ARMY  AND  NAVY. 

Bernadou's  Smokeless  Powder,  Nitro-cellulose,  and  theJTheory  of  the  Cellulose 

Molecule i2mo,  2  50 

*  Bruff's  Text-book  Ordnance  and  Gunnery 8vo,  6  oo 

Chase's  Screw  Propellers  and  Marine  Propulsion 8vo,  3  oo 

Craig's  Azimuth 4to,  3  50 

Cre  lore  and  Squire's  Polarizing  Photo-chronograph 8vo,  3  oo 

Cronkhite's  Gunnery  for  Non-commissioned  Officers 241110..  morocco,  2  oo 

*  Davis's  Elements  of  Law 8vo,  2  50 

*  Treatise  on  the  Military  Law  of  United  States 8vo,    7  oo 

Sheep,  7  50 

De  Brack's  Cavalry  Outpost  Duties.     (Carr.) 24010  morocco,    2  oo 

Dietz's  Soldier's  First  Aid  Handbook i6mo,  morocco,    i  25 

*  Dredge's  Modern  French  Artillery 4to,_half  morocco,    15  oo 

Durand's  Resistance  and  Propulsion  of  Ships 8vo,    5  oo 

*  Dyer's  Handbook  of  Light  Artillery i2mo,    3  oo 

Eissler's  Modern  High  Explosives 8vo,    4  oo 

*  Fiebeger's  Text-book  on  Field  Fortification SmaU^Svo,    2  oo 

Hamilton's  The  Gunner's  Catechism i8mo,    i  oo 

*  Hoff's  Elementary  Naval  Tactics 8vo,    i  30 

Ingalls's  Handbook  of  Problems  in  Direct  Fire 8vo,    4  oo 

*  Ballistic  Tables 8vo,    i  50 

*  Lyons's  Treatise  on  Electromagnetic  Phenomena.   Vols.  I.  and  II .  .  8vo,  each,'!  6  op 

*  Mahan's  Permanent  Fortifications.     (Mercur.) 8vo,  half  morocco,    7  50 

Manual  for  Courts-martial i6mo>  morocco,    i  50 

*  Mercur's  Attack  of  Fortified  Places i2mo,    2  oo 

*  Elements  of  the  Art  of  War 8vo,    4  oo 

Metcalf 's  Cost  of  Manufactures — And  the  Administration^  Workshops,  Public 

and  Private 8vo,    5  oo 

*  Ordnance  and  Gunnery i2mo,    5  oo 

Murray's  Infantry  Drill  Regulations i8mo,fpaper,        10 

*  Phelps's  Practical  Marine  Surveying 8vo,    2  50 

Powell's  Army  Officer's  Examiner 121110,    4  oo 

Sharpe's  Art  of  Subsisting  Armies  in  War i8mo,  morocco,    i  50 

2 


*  Walke's  Lectures  on  Explosives 8vo,  4  oo 

*  Wheeler's  Siege  Operations  and  Military  Mining 8vo,  2  oo 

Winthrop's  Abridgment  of  Military  Law i2mo,  2  30 

Woodhull's  Notes  on  Military  Hygiene i6mo,  i  50 

Young's  Simple  Elements  of  Navigation i6mo.  morocco,  i  oo 

Second  Edition,  Enlarged  and  Revised i6mo,  morocco,  2  •« 


ASSAYING. 

Fletcher's  Practical  Instructions  in  Quantitative  Assaying  with  the  Blowpipe. 

i2mo,  morocco,  i  50 

Furman's  Manual  of  Practical  Assaying 8vo,  3  oo 

Miller's  Manual  of  Assaying I2mo,  I  oo 

O'Driscoll's  Notes  on  the  Treatment  of  Gold  Ores 8vo.  2  oo 

Ricketts  and  Miller's  Notes  on  Assaying 8vo,  3  oo 

Hike's  Modern  Electrolytic  Copper  Refining 8vo,  3  oo 

Wilson's  Cyanide  Processes i2mo,  i  50 

Chlorination  Process i2mo,  i  50 


ASTRONOMY. 

Comstock's  Field  Astronomy  for  Engineers 8vo,  2  50 

Craig's  Azimuth 4to,  3  50 

Doolittle's  Treatise  on  Practical  Astronomy 8vo,  4  oo 

Gore's  Elements  of  Geodesy 8vo,  2  90 

Hayford's  Text-book  of  Geodetic  Astronomy 8vo,  3  oo 

Merriman's  Elements  of  Precise  Surveying  and  Geodesy 8vo,  2  50 

*  Michie  and  Harlow's  Practical  Astronomy 8vo,  3  oo 

*  White's  Elements  of  Theoretical  and  Descriptive  Astronomy lamo,  2  oo 

BOTANY. 

Davenport's  Statistical  Methods,  with  Special  Reference  to  Biological  Variation. 

i6mo,  morocco,  i  25 

Thome  and  Bennett's  Structural  and  Physiological  Botany i6mo,  2  25 

Westermaier's  Compendium  of  General  Botany.     (Schneider.) 8vo,  2  oo 

CHEMISTRY. 

Adriance's  Laboratory  Calculations  and  Specific  Gravity  Tables lamo,  /  25 

Allen's  Tables  for  Iron  Analysis 8ro,  3  oo 

Arnold's  Compendium  of  Chemistry.     (Mandel.)     (In  preparation.) 

Austen's  Notes  for  Chemical  Students 12010,  I  50 

Bernadou's  Smokeless  Powder. — Nitro-cellulose,  and  Theory  of  the  Cellulose 

Molecule i2mo,  2  50 

Bolton's  Quantitative  Analysis 8vo,  i  50 

*  Browning's  Introduction  to  the  Rarer  Elements 8vo,  I  50 

Brush  and  Penfield's  Manual  of  Determinative  Mineralogy 8vo,  4  oo 

Classen's  Quantitative  Chemical  Analysis  by  Electrolysis.  (Bolt wood.)  . . .   8vo,  3  oo 

Cohn's  Indicators  and  Test-papers I2mo,  2  oo 

Tests  and  Reagents , 8vo,  3  oo 

Copeland's  Manual  of  Bacteriology.     (In  preparation.) 

Craft's  Short  Course  in  Qualitative  Chemical  Analysis.  (Schaeffer.) 12 mo,  i  90 

Drechsel's  Chemical  Reactions.     (Merrill  ) I2mo,  i  25 

Ihihem's  Thermodynamics  and  Chemistry.     (Burgess.) STO,  4  oo 

Eissler's  Modern  High  Explosives 8vo,  4  oo 

3 


Effront's  Enzymes  and  their  Applications.     (Prescott.) 8vo,  3  oo 

Erdmann's  Introduction  to  Chemical  Preparations.     (Dunlap.) i2mo,  i   25 

Fletcher's  Practical  Instructions  in  Quantitative  Assaying  with  the  Blowpipe. 

1 2mo,  morocco,  i   50 

Fowler's  Sewage  Works  Analyses i2mo,  2  oo 

Fresenius's  Manual  of  Qualitative  Chemical  Analysis.     (Wells.) 8vo,  5  oo 

Manual  of  Qualitative  Chemical  Analysis.     Parti.    Descriptive.     (Wells.) 

8vo,  3  oo 

System   of   Instruction   in    Quantitative    Chemical   Analysis.      (Cohn.) 
2  vols.     (Shortly.) 

Fuertes's  Water  and  Public  Health i2mo,  i  50 

Furman's  Manual  of  Practical  Assaying 8vo,  3  oo 

Gill's  Gas  and  Fuel  Analysis  for  Engineers i2mo,  i  25 

Grotenfelt's  Principles  of  Modern  Dairy  Practice.     (Wo  11.) i2mo,  2  ob 

Hammarsten's  Text-book  of  Physiological  Chemistry.     (Mandel.) 8vo,  4  oo 

Helm's  Principles  of  Mathematical  Chemistry.     (Morgan.) i2mo,  i  50 

Hinds's  Inorganic  Chemistry 8vo,  3  oo 

*  Laboratory  Manual  for  Students I2mo,  75 

Holleman's  Text-book  of  Inorganic  Chemistry.     (Cooper.) 8vo,  2  50 

Text-book  of  Organic  Chemistry.     (Walker  and  Mott.) 8vo,  2  so 

Hopkins's  Oil-chemists'  Handbook 8vo,  3  oo 

Jackson's  Directions  for  Laboratory  Work  in  Physiological  Chemistry.  .8vo,  i  oo 

Keep's  Cast  Iron 8vo,  2  50 

Ladd's  Manual  of  Quantitative  Chemical  Analysis . i2mo  i  oo 

Landauer's  Spectrum  Analysis.    (Tingle.) 8vo,  3  oo 

Lassar-Cohn's  Practical  Urinary  Analysis.     (Lorenz.) i2mo,  i  oo 

Leach's  The  Inspection  and  Analysis  of  Food  with  Special  Reference  to  State 

Control.     (In  preparation.) 

Lwb's  Electrolysis  and  Electrosynthesis  of  Organic  Compounds.  (Lorenz.)  i2mo,  i  oo 

Mandel's  Handbook  for  Bio-cherrical  Laboratory i2mo,  i   so 

*  Martin's  Laboratory  Guide  to  Qualitative  Analysis  with  the  Blowpipe .  .  12 mo,  60 
Mason's  Water-supply.     (Considered  Principally  from  a  Sanitary  Standpoint.) 

3d  Edition,  Rewritten 8vo,  4  oo 

Examination  of  Water.  (Chemical  and  Bacteriological.) i2mo,  i  25 

Meyer's  Determination  of  Radicles  in  Carbon  Compounds.  (Tingle.).  .  i2mo,  i  oo 

Miller's  Manual  of  Assaying I2mo,  i  oo 

Mixter's  Elementary  Text-book  of  Chemistry I2mo,  i  50 

Morgan's  Outline  of  Theory  of  Solution  and  its  Results I2mo,  i  oo 

Elements  of  Physical  Chemistry i2mo,  2  oo 

Nichols's  Water-supply.  (Considered  mainly  from  a  Chemical  and  Sanitary 

Standpoint,  1883.) 8vo,  2  50 

O'Brine's  Laboratory  Guide 'in  Chemical  Analysis 8vo,  2  oo 

O'Driscoll's  Notes  on  the  Treatment  of  Gold  Ores 8vo,  2  oo 

Ost  and  Kolbeck's  Text-book  of  Chemical  Technology.  (Lorenz — Bozart.) 

(In  preparation.) 

*  Penfield's  Notes  on  Determinative  Mineralogy  and  Record  of  Mineral  Tests. 

8vo,  paper,  50 

Pictet's   The   Alkaloids   and   their   Chemical  Constitution.      (Biddle.)      (In 
preparation.) 

Pinner's  Introduction  to  Organic  Chemistry.     (Austen.) i2mo,  i  50 

Poole's  Calorific  Power  of  Fuels 8vo,  3  oo 

*  Reisig's  Guide  to  Piece-dyeing 8vo,  25  oo 

Richards  and  Woodman's  Air  .Water,  and  Food  from  a  Sanitary  Standpoint.  8vo,  2  oo 

Richards's  Cost  of  Living  as  Modified  by  Sanitary  Science i2mo,  i  oo 

Cost  of  Food  a  Study  in  Dietaries I2mo,  i  oo 

*  Richards  and  Williams's  The  Dietary  Computer 8vo,  i  50 

Ricketts  and  Russell's  Skeleton  Notes  upon  Inorganic  Chemistry.     (Part  I. — 

Non-metallic  Elements.) 8vo,  morocco,  75 

4 


Ricketts  and  Miller's  Notes  on  Assaying 8vo,  3  oo 

Rideal's  Sewage  and  the  Bacterial  Purification  of  Sewage ^.8vo,  3'  30 

Ruddiman's  Incompatibilities  in  Prescriptions 8vo,  a  OO 

Salkowski's  Physiological  and  Pathological  Chemistry.     (Orndorff.) 

(Shortly.) 

Schimpf  s  Text-book  of  Volumetric  Analysis izmo,  50 

Essentials  of  Volumetric  Analysis izmo,  25 

^pencer's  Handbook  for  Chemists  of  Beet-sugar  Houses i6mo,  morocco,  oo 

Handbook  for  Sugar  Manufacturers  and  their  Chemists. .  i6mo,  morocco,  oo 

Stockbndge's  Rocks  and  Soils 8vo,  50 

*  Tillman's  Elementary  Lessons  in  Heat 8vo,  50 

*  Descriptive  General  Chemistry 8vo  3  oo 

Treadwell's  Qualitative  Analysis.     (HalL) 8vo,  3  oo 

Turneaure  and  Russell's  Public  Water-supplies 8vo,  5  oo 

Van  Deventer's  Physical  Chemistry  for  Beginners.     (Boltwood.) i2mo,  i  50 

*  Walke's  Lectures  on  Explosives 8vo,  4  oo 

Wells's  Laboratory  Guide  in  Qualitative  Chemical  Analysis. 8vo,  i  50 

Short  Course  in  Inorganic  Qualitative  Chemical  Analysis  for  Engineering 

Students i2mo,  i  50 

Whipple's  Microscopy  of  Drinking-water 8vo,  3  50 

Wiechmann's  Sugar  Analysis Small  8vo.  2  5» 

Wilson's  Cyanide  Processes xamo,  i  30 

Chlorination  Process I2mo  i  50 

Wulling's  Elementary  Course  in  Inorganic  Pharmaceutical  and  Medical  Chem- 
istry.    I2mo,  2  oo 

CIVIL  ENGINEERING. 

BRIDGES  AND    ROOFS.       HYDRAULICS       MATERIALS   OF  ENGINEERING. 
RAILWAY   ENGINEERING. 

Baker's  Engineers'  Surveying  Instruments i2mo,  3  oo 

Bixby's  Graphical  Computing  Table Paper  1 0,^X24}  inches.  25 

**  Burr's  Ancient  and  Modern  Engineering  and  the  Isthmian  CflnaL     (Postage, 

27  cents  additional.) 8vo,  net,  3  50 

Comstock's  Field  Astronomy  for  Engineers 8vo,  2  50 

Davis's  Elevation  and  Stadia  Tables 8vo,  i  oo 

Elliott's  Engineering  for  Land  Drainage 1 2mo,  i  50 

Practical  Farm  Drainage i amo,  i  oo 

Folwell's  Sewerage.     (Designing  and  Maintenance.) 8vo,  3  oo 

Freitag's  Architectural  Engineering.     2d  Edition,  Rewritten 8vo,  3  50 

French  and  Ives's  Stereotomy 8vo,  2  50 

Goodhue's  Municipal  Improvements I2mo,  I  75 

Goodrich's  Economic  Disposal  of  Towns'  Refuse 8vo,  3  50 

Gore's  Elements  of  Geodesy   8vo,  a  50 

Hayford's  Text-book  of  Geodetic  Astronomy 8vo,  3  oo 

Howe's  Retaining  Walls  for  Earth i2mo,  i  25 

Johnson's  Theory  and  Practice  of  Surveying Small  8vo,  4  oo 

Statics  by  Algebraic  and  Graphic  Methods .-• 8vo,  2  oo 

Kiersted's  Sewage  Disposal I2mo,  i  25 

Laplace's  Philosophical  Essay  on  Probabilities.     (Truscott  and  Emory.)  i2mo,  2  oo 

Mahan's  Treatise  on  Civil  Engineering.     (1873  )     (Wood.) 8vo,  5  oo 

*  Descriptive  Geometry    8vo,  i   50 

Merriman's  Elements  of  Precise  Surveying  and  Geodesy 8vo,  2  50 

Elements  of  Sanitary  Engineering 8vo,  2  oo 

Merriman  and  Brooks'*?  Handbook  for  Surveyors i6mo,  morocco,  2  oo 

Nugent's  Plane  Surveying 8vo,  3  50 

Ogden's  Sewer  Design I2mo,  2  oo 

Patton's  Treatise  on  Civil  Engineering 8vo  half  leather,  7   50 

5 


Reed's  Topographical  Drawing  and  Sketching 4to,  5  oo 

Rideal's  Sewage  and  the  Bacterial  Purification  of  Sewage 8vo,  3  50 

Siebert  and  Biggin's  Modern  Stone-cutting  and  Masonry .8vo,  i  50 

Smith's  Manual  of  Topographical  Drawing.     (McMillan.) 8vo,  2  50 

Sondericker's   Graphic   Statics,   witn   Applications   to   Trusses.  Beams,   and 

Arches 8vo,  2  oo 

*  Trautwine's  Civil  Engineer's  Pocket-book i6mo,  morocco,  5  oo 

Wait's  Engineering  and  Architectural  Jurisprudence 8vo,  6  oo 

Sheep,  6  50 

Law  of  Operations  Preliminary  to  Construction  in  Engineering  and  Archi- 
tecture  8vo,  5  oo 

Sheep,  5  50 

Law  of  Contracts 8vo,  3  oo 

Warren's  Stereotomy — Problems  in  Stone-cutting 8vo,  2  50 

Webb's  Problems  in  the  U«?e  and  Adjustment  of  Engineering  Instruments. 

i6mo,  morocco,  i  25 

*  Wheeler's  Elementary  Course  of  Civil  Engineering 8vo,  4  oo 

Wilson's  Topographic  Surveying 8vo,  3  50 


BRIDGES  AND  ROOFS. 

Bailer's  Practical  Treatise  on  the  Construction  of  Iron  Highway  Bridges.  .8vo,  2  oo 

*         Thames  River  Bridge 4to,  paper,  5  oo 

Burr's  Course  on  the  Stresses  in  Bridges  and  Roof  Trusses,  Arched  Ribs,  and 

Suspension  Bridges 8vo,  3  50 

»u  Bois's  Mechanics  of  Engineering.     VoL  II Small  4to,    10  oo 

Foster's  Treatise  on  Wooden  Trestle  Bridges 4to,  5  oo 

Fowler's  Coffer-dam  Process  for  Piers 8vo,  2  50 

Greene's  Roof  Trusses 8vo,  i  25 

Bridge  Trusses 8vo,  2  50 

Arches  in  Wood,  Iron,  and  Stone 8vo,  2  50 

Howe's  Treatise  on  Arches 8vo  4  oo 

Design  of  Simple  Roof-trusses  in  Wood  and  Steel 8vo,  2  oo 

Johnson,  Bryan,  and  Turneaure's  Theory  and  Practice  in  the  Designing  of 

Modern  Framed  Structures Small  4to,  10  oo 

Merriman  and  Jacoby's  Text-book  on  Roofs  and  Bridges: 

Part  I. — Stresses  in  Simple  Trusses 8vo,  2  50 

Part  H.— Graphic  Statics 8vo,  2  50 

Part  III.— Bridge  Design.     4th  Edition,  Rewritten 8vo,  2  50 

Part  IV.— Higher  Structures 8vo,  2  50 

Morison's  Memphis  Bridge 4to,  10  oo 

Waddell's  De  Pontibus,  a  Pocket-book  for  Bridge  Engineers. .  .  i6mo,  morocco,  3  oo 

Specifications  for  Steel  Bridges i2mo,  i  25 

Wood's  Treatise  on  the  Theory  of  the  Construction  of  Bridges  and  Roofs.Svo,  2  oo 
Wright's  Designing  of  Draw-spans: 

Part  I.  — Plate-girder  Draws 8vo,  2  50 

Part  II. — Rivetedhtruss  and  Pin-connected  Long-span  Draws 8vo,    2  50 

Two  parts  in  one  volume 8vo,  3  50 


HYDRAULICS. 

Bazin's  Experiments  upon  the  Contraction  of  the  Liquid  Vein  Issuing  from  an 

Orifice.     (Trautwine.) 8vo,  2  oo 

B0vey*s  Treatise  on  Hydraulics 8vo,  5  oo 

Church's  Mechanics  of  Engineering 8vo,  6  oo 

Diagrams  of  Mean  Velocity  of  Water  in  Open  Channels paper,  i  50 

6 


Coffin's  Graphical  Solution  of  Hydraulic  Problems i6mo,  morocco,  2  50 

Flather's  Dynamometers,  and  the  Measurement  of  Power i2mo,  3  oo 

Folwell's  Water-supply  Engineering 8vo,  4  oo 

Ffizell's  Water-power 8vo,  5  oo 

Fuertes's  Water  and  Public  Health i2mo,  i  50 

Water-filtration  Works iamo,  2  50 

Ganguillet  and  Kutter's  General  Formula  for  the  Uniform  Flow  of  Water  in 

Rivers  and  Other  Channels.     (Hering  and  Trau twine.) 8vo,  4  oo 

Hazen's  Filtration  of  Public  Water-supply 8vo,  3  oo 

Hazlehurst's  Towers  and  Tanks  for  Water-works 8vo,  2  50 

Herschel's  115  Experiments  on  the  Carrying  Capacity  of  Large,  Riveted,  Metal 

Conduits 8vo,  2  oo 

Mason's    Water-supply.     (Considered    Principally   from   a    Sanitary   Stand- 
point.)    3d  Edition,  Rewritten 8vo,  4  oo 

Merriman's  Treatise  on  Hydraulics,     gth  Edition,  Rewritten 8vo,  5  oo 

*  Michie's  Elements  of  Analytical  Mechanics 8vo,  4  oo 

Schuyler's   Reservoirs  for  Irrigation,   Water-power,  and   Domestic   Water- 
supply I Large  8vo,  5  oo 

•*  Thomas  and  Watt's  Improvement  of  Riyers.     (Post.,  44  c.  additional),  4to,  6  oo 

Tumeaure  and  Russell's  Public  Water-supplies 8vo,  5  oo 

Wegmann's  Desiem  and  Construction  of  Dams 4to,  5  oo 

Water-supp'y  of  the  City  of  New  York  from  1658  to  1895 4to,  10  oo 

Weisbach's  Hvtiraulics  and  Hydraulic  Motors.     (Du  Bois.) 8vo,  5  oo 

Wilson's  Manual  of  Irrigation  Engineering Small  8vo.  4  oo 

Wolff's  Windmill  as  a  Prime  Mover 8vo,  3  oo 

Wood's  Turbines 8vo,  2  50 

Elements  of  Analytical  Mechanics 8vo,  3  oo 


MATERIALS  OF  ENGINEERING. 

Baker's  Treatise  on  Masonry  Construction 8vo,  5  oo 

Roads  and  Pavements 8vo,  5  oo 

Black's  United  States  Public  Works Oblong  4to,  5  oo 

Bovey's  Strength  of  Materials  and  Theory  of  Structures 8vo,  7  SO 

Burr's,  Elasticity  and  Resistance  of  the  Materials  of  Engineering.     6th  Edi- 
tion, Rewritten 8vo,  7  50 

Byrne's  Highway  Construction 8vo.  5  oo 

Inspection  of  the  Materials  and  Workmanship  Employed  in  Construction. 

i6mo,  3  oo 

Church's  Mechanics  of  Engineering 8vo,  6  oo 

Du  Bois's  Mechanics  of  Engineering.     VoL  I Small  4to,  7  50 

Johnson's  Material^  of  Construction Large  8vo,  6  oo 

Keep's  Cast  Iron 8vo,  2  50 

Lanza's  Applied  Mechanics 8vo,  7  50 

Marte ns's  Handbook  on  Testing  Materials.     (Henning.)     2>ols. 8vo,  750 

Merrill'!.  Stones  for  Building  and  Decoration 8vo,  5  oo 

Merriman's  Text-book  on  the  Mechanics  of  Materials 8vo,  4  oo 

Strength  of  Materials I2mo,  i  oo 

Metcalf's  Steel     A  Manual  for  Steel-users i2mo,  2  oo 

Patton's  Practical  Treatise  on  Foundations 8vo,  5  oo 

Rockwell's  Roads  and  Pavements  in  France i2mo,  i  25 

Smith's  Wire :  Its  Use  and  Manufacture Small  4to,  3  oo 

Materials  of  Machines 1 2mo,  i  oo 

Snow's  Principal  Species  of  Wood 8vo,  3  50 

Spalding's  Hydraulic  Cement i2mo,  2  oo 

Text-book  on  .Roads  and  Pavements i2mo,  2  oo 

7 


Thurston's  Materials  of  Engineering.     3  Parts : 8vo,  8  oo 

Part  I. — Non-metallic  Materials  of  Engineering  and  Metallurgy 8vo,  2  oo 

Part  II. — Iron  and  Steel 8vo,  3  50 

Part  III. — A  Treatise  on  Brasses,  Bronzes,  and  Other  Alloys  and  their 

Constituents 8vo,  2  50 

Fhurston's  Text-book  of  the  Materials  of  Construction 8vo,  5  oo 

Pillson's  Street  Pavements  and  Paving  Materials 8vo,  4  oo 

Waddell's  De  Pontibus.     (A  Pocket-book  for  Bridge  Engineers.) . .  i6mo,  mor.,  3  oo 

Specifications  for  Steel  Bridges 1 21x10 ,  i   25 

Wood's  Treatise  on  the  Resistance  of  Materials,  and  an  Appendix  on  the  Pres- 
ervation of  Timber     8vo,  2  oo 

Elements  of  Analytical  Mechanics 8vo,  3  oo 

Wood's  Rustless  Coatings.     (Shortly.) 


RAILWAY  ENGINEERING. 

Andrews's  Handbook  for  Street  Railway  Engineers.     3X5  inches,  morocco,  i  25 

Berg's  Buildings  and  Structures  of  American  Railroads 4to,  5  oo 

Brooks's  Handbook  of  Street  Railroad  Location i6mo  morocco,  i  50 

Butts's  Civil  Engineer's  Field-book i6mo,  morocco,  2  50 

Crandall's  Transition  Curve i6mo,  morocco,  i  50 

Railway  and  Other  Earthwork  Tables *. . .  .8vo,  i  50 

Dawson's  "Engineering"  and  Electric  Traction  Pocket-book.    i6mo,  morocco,  5  oo 

Dredge's  History  of  the  Pennsylvania  Railroad:    (1879) Paper,  5  oo 

*  Drinker's  Tunneling,  Explosive  Compounds,  and  Rock  Drills,  4to,  half  mor.,    25  oo 

Fisher's  Table  of  Cubic  Yards ; Cardboard  25 

Godwin's  Railroad  Engineers'  Field-book  and  Explorers'  Guide i6mo,  mor.,  2   50 

Howard's  Transition  Curve  Field-book i6mo,  morocco,  i  50 

Hudson's  Tables  for  Calculating  the  Cubic  Contents  of  Excavations  and  Em- 
bankments  8vo,  i   oo 

Molitor  and  Beard's  Manual  for  Resident  Engineers i6mo,  i   oo 

Nagle's  Field  Manual  for  Railroad  Engineers i6mo,  morocco.  3  oo 

Philbrick's  Field  Manual  for  Engineers i6mo,  morocco,  3  oo 

Pratt  and  Alden's  Street-railway  Road-bed 8vo,  2  oo 

Searles's  Field  Engineering i6mo,  morocco,  3  oo 

Railroad  Spiral i6mo,  morocco,  i   50 

Taylor's  Prismoidal  Formulae  and  Earthwork 8vo,  i  50 

*  Trautwine's  Method  of  Calculating  the  Cubic  Contents  of  Excavations  and 

Embankments  by  the  Aid  of  Diagrams 8vo,  2  oo 

The  Field  Practice  of  tLaying    Out    Circular    Curves    for    Railroads. 

i2ino,  morocco,  2   50 

*  Cross-section  Sheet Paper,  25 

Webb's  Railroad  Construction.     2d  Edition,  Rewritten i6mo.  morocco,  5  oo 

Wellington's  Economic  Theory  of  the  Location  of  Railways Small  8vo,  5  oo 


DRAWING. 

Barr's  Kinematics  of  Machinery 8vo,  2  50 

*  Bartlett's  Mechanical  Drawing 8vo,  3  oo 

*  "  "  "         Abridged  Ed 8vo,  i  50 

Coolidge's  Manual  of  Drawing 8vo,  paper,  i   oo 

Durley's  Kinematics  of  Machines 8vo,  4  oo 

Hill's  Text-book  on  Shades  and  Shadows,  and  Perspective 8vo,  2  oo 

Jones's  Machine  Design: 

Part  I. — Kinematics  of  Machinery 8vo,  i  50 

Part  II. — Form,  Strength,  and  Proportions  of  Parts 8vo,  3  oo 


MacCord's  Elements  of  Descriptive  Geometry 8vo,  3  oo 

•Kinematics;  or,  Practical  Mechanism 8vo,  5  oo 

Mechanical  Drawing 4to,  4  oo 

Velocity  Diagrams 8vo,  i  50 

*  Mahan's  Descriptive  Geometry  and  Stone-cutting 8vo,  i  50 

Industrial  Drawing.  (Thompson.) 8vo,  3  5<> 

Reed's  Topographical  Drawing  and  Sketching 4to,  5  oo 

Reid's  Course  in  Mechanical  Drawing 8vo,  2  oo 

Text-book  of  Mechanical  Drawing  and  Elementary  Machine  Design.  .8vo.  3  oo 

Robinson's  Principles  of  Mechanism 8vo,  3  oo 

Smith's  Manual  of  Topographical  Drawing.  (McMillan.) 8vo,  2  50 

Warren's  Elements  of  Plane  and  Solig  Free-hand  Geometrical  Drawing. .  I2mo, 


Drafting  Instruments  and  Operations I2mo, 

Manual  of  Elementary  Projection  Drawing   I2mo, 

Manual  of  Elementary  Problems  in  the  Linear  Perspective  of  Form  and 


Shadow i2mo,  oo 

Plane  Problems  in  Elementary  Geometry i2mo,  25 

Primary  Geometry I2mo,  75 

Elements  of  Descriptive  Geometry,  Shadows,  and  Perspective 8vo,  3  5° 

General  Problems  of  Shades  and  Shadows             8vo,  3  oo 

Elements  of  Machine  Construction  and  Drawing 8vo,  7  So 

Problems.  Theorems,  and  Examples  in  Descriptive  Geometry 8vo,  2  50 

Weisbach's  Kinematics  and  the  Power  of  Transmission.       v Hermann  and 

Klein.) 8vo,  5  oo 

Whelpley's  Practical  Instruction  in  the  Art  of  Letter  Engraving I2mo,  2  oo 

Wilson's  Topographic  Surveying 8vo,  3  50 

Free-hand  Perspective 8vo,  2  50 

Free-hand  Lettering 8vo,  i  oo 

Woo  If 's  Elementary  Course  in  Descriptive  Geometry Large  8vo,  3  oo 

ELECTRICITY  AND   PHYSICS. 

Anthony  and  Brackett's  Text-book  of  Physics.     (Magie.) Small  8vo,  3  oo 

Anthony's  Lecture-notes  on  the  Theory  of  Electrical  Measurements 12 mo,  i  oo 

Benjamin's  History  of  Electricity 8vo,  3  oo 

Voltaic  Cell 8vo,  3  oo 

Classen's  Quantitative  Chemical  Analysis  by  Electrolysis.    (Boltwood.).  .8vo,  3  oo 

Crehore  and  Sauier's  Polarizing  Photo-chronograph 8vo.  3  oo 

Dawson's  "Engineering"  and  Electric  Traction  Pocket-book.  .i6mo,  morocco,  5  oo 
Dolezalek's    Theory    of    the    Lead    Accumulator.     (Storage  Battery.) 
(Shortly.)     (Von  Ende.) 

Duhem's  Thermodynamics  and  Chemistry.     (Burgess.) 8vo,  4  oo 

blather's  Dvnamo meters,  and  the  Measurement  of  Power 1 2010,  3  oo 

Giioerrs  De  Magnete.     (Mottelay.) 8vo,  2  50 

Hanchett's  Alternating  Currents  Explained.     (Shortly.) 

Holman's  Precision  of  Measurements 8vo,  2  oo 

Telescopic  Mirror-scale  Method,  Adjustments,  and  Tests Large  JJvo,  75 

Landauer's  Spectrum  Analysis.    (Tingle.) 8vo,  3  OO 

Le  Chatelier's  High-temperature  Measurements.  (Boudouard — Burgess.  }i2mo,  3  oo 

Lob's  Electrolysis  and  Electrosynthesis  of  Organic  Compounds.  (Lorenz.)  i2mo,  i  oo 

*  Lyons's  Treatise  on  Electromagnetic  Phenomena.     Vols.  I.  and  II.  8vo,  each,  6  oo 

*  Michie.     Elements  of  Wave  Motion  Relating  to  Sound  and  Light 8vo,  4  oo 

Niaudet's  Elementary  Treatise  on  Electric  Batteries.     (Fishoack. ) i2mo,  2  50 

*  Parshall  and  Hobart's  Electric  Generators Small  4to.  half  morocco,  10  oo 

*  Rosenberg's  Electrical  Engineering.    (Haldane  Gee — Kinzbrunner.).  .  .  .8vo,  i   50 

Ryan,  Norris,  and  Hoxie's  Electrical  Machinery.     Vol.  1 8vo,  2  50 

Thurston's  Stationary  Steam-engines 8vo,  2  50 

*  Tillman's  Elementary  Lessons  in  Heat 8vo,  i   50 

9 


Tory  and  Pitcher's  Manual  of  Laboratory  Physics Small  8vo,  2  oo 

Hike's  Modern  Electrolytic  Copper  Refining 8vo,  3  oo 

LAW. 

*  Davis's  Elements  of  Law 8vo,  2  50 

*  Treatise  on  the  Military  Law  of  United  States 8vo,  7  oo 

*  Sheep,  7  SO 

Manual  for  Courts-martial i6mo,  morocco,  i  50 

Wait's  Engineering  and  Architectural  Jurisprudence 8vo,  6  oo 

Sheep,  6  50 

Law  of  Operations  Preliminary  to  Construction  in  Engineering  and  Archi- 
tecture      8vo,  5  oo 

Sheep,  5  50 

Law  of  Contracts 8vo,  3  oo 

Winthrop's  Abridgment  of  Military  Law i2mo,  2  50 

MANUFACTURES. 

Bernadou's  Smokeless  Powder — Nitro-cellulose  and  Theory  of  the  Cellulose 

Molecule i2mo,  z  50 

Holland's  Iron  Founder i2mo,  2  50 

"  The  Iron  Founder,"  Supplement i2mo,  2  50 

Encyclopedia  of  Founding  and  Dictionary  of  Foundry  Terms  Used  in  the 

Practice  of  Moulding i2mo,  3  oo 

Eissler's  Modern  High  Explosives 8vo,  4  oo 

Effront's  Enzymes  and  their  Applications.     (Prescott.) 8vo,  3  oo 

Fitzgerald's  Boston  Machinist i8mo,  i  oo 

Ford's  Boiler  Making  for  Boiler  Makers i8mo,  i  oo 

Hopkins's  Oil-chemists'  Handbook 8vo,  3  oo 

Keep's  Cast  Iron 8vo,  a  50 

Leach's  The  Inspection  and  Analysis  of  Food  with  Special  Reference  to  State 

Control.     (In  preparation.) 

Metcalf's  Steel.     A  Manual  for  Steel-users i2mo,  2  oo 

Metcalfe's  Cost  of  Manufactures —And  the  Administration    of  Workshops. 

Public  and  Private 8vo,  5  oo 

Meyer's  Modern  Locomotive  Construction 4to,  10  oo 

*  Reisig's  Guide  to  Piece-dyeing 8vo,  25  oo 

Smith's  Press-working  of  Metals 8vo,  3  oo 

Wire :  Its  Use  and  Manufacture Small  4to,  3  oo 

Spalding's  Hydraulic  Cement i2mo,  2  oo 

Spencer's  Handbook  for  Chemists  of  Beet-sugar  Houses i6mo,  morocco,  3  oo 

Handbook  tor  sugar  Manufacturers  and  their  Chemists..  .  i6mo,  morocco,  2  oo 
Thurston's  Manual  of  Steam-boilers,  their  Designs,  Construction  and  Opera- 
tion   8vo,  s  eo 

*  Walke's  Lectures  on  Explosives 8vo,    4  oo 

West's  American  Foundry  Practice 121110,    2  50 

Moulder's  Text-book i2mo,  2  50 

Wiechmann's  Sugar  Analysis Small  8vo,  2  50 

Wolff's  Windmill  as  a  Prime  Mover 8vo,  3  oo 

Woodbury's  Fire  Protection  of  Mills 8vo,  2  50 

MATHEMATICS. 

Baker's  Elliptic  Functions 8vo,    i  50 

^Bass's  Elements  of  Differential  Calculus i2mo,    4  oo 

Briggs's. Elements 'of  Plane  Analytic  Geometry i2mo,  i  oo 

10 


So 
50 
So 
25 

75 
50 


Compton's  Manual  of  Logarithmic  Computations I2mo, 

Davis's  Introduction  to  the  Logic  of  Algebra 8vo, 

*  Dickson's  College  Algebra   Large  I2mo, 

*  Introduction  to  the  Theory  of  Algebraic  Equations   Large  izmo, 

Halsted's  Elements  of  Geometry   8vo, 

Elementary  Synthetic  Geometry 8vo. 

Rational  Geometry.     (Shortly.') 

*  Johnson's  Three-place  Logarithmic  Tables:    Vest-pocket  size paper,        15 

100  copies  for    5  oo 

*  Mounted  on  heavy  cardboard,  8  X 10  inches,         25 

10  copies  for    2  oo 

Elementary  Treatise  on  the  Integral  Calculus Small  8vo,    i  50 

Curve  Tracing  in  Cartesian  Co-ordinates I2mo,    i  oo 

Treatise  on  Ordinary  and  Partial  Differential  Equations Small  8vo,    3  50 

Theory  of  Errors  and  the  Method  of  Least  Squares I2mo,    i  50 

*  Theoretical  Mechanics i2mo,    3  oo 

Laplace's  Philosophical  Essay  on  Probabilities.     (Truscott  and  Emory.)  i2mo,    200 

*  Ludlow  and  Bass.     Elements  of  Trigonometry  and  Logarithmic  and  Other 

Tables 8vo,    3  oo 

Trigonometry  and  Tables  published  separately Each,    2  oo 

Maurer's  Technical  Mechanics 8vo,     4  o« 

Merriman  and  Woodward's  Higher  Mathematics 8vo,    5  oo 

Merriman's  Method  of  Least  Squares 8vo,    a  oo 

Rice  and  Johnson's  Elementary  Treatise  on  the  Differential  Calculus .  Sm.,  8vo,    3  oo 

Differential  and  Integral  Calculus.     2  vols.  in  one Small  8vo,    2  50 

Wood's  Elements  of  Co-ordinate  Geometry 8vo,    2  oo 

Trigonometry:  Analytical,  Plane,  and  Spherical.  , i2mo,    I  oo 


MECHANICAL   ENGINEERING. 
MATERIALS  OF  ENGINEERING,  STEAM-ENGINES  AND  BOILERS. 

Baldwin's  Steam  Heating  for  Buildings I2mo,  2  50 

Barr's  Kinematics  of  Machinery 8vo,  2  50 

•  Bartlett's  Mechanical  Drawing 8vo,  3  oo 

*  "                 "               "        Abridged  Ed 8vo,  i  5* 

Benjamin's  Wrinkles  and  Recipes i2mo,  2  oo 

Carpenter's  Experimental  Engineering 8vo,  6  oo 

Heating  and  Ventilating  Buildings 8vo,  4  oo 

Gary's  Smoke  Suppression  in  Plants  using  Bituminous  CoaL      (In  prep- 
aration.') 

Clerk's  Gas  and  Oil  Engine. . .  * Small  8vo,  4  oo 

Coolidge's  Manual  of  Drawing 8vo,    paper,  I  oo 

Cromwell's  Treatise  on  Toothed  Gearing 121110,  I  50 

Treatise  on  Belts  and  Puheys I2mo,  i  50 

Durley's  Kinematics  of  Machines 8vo,  4  oo 

Flather's  Dynamometers  and  the  Measurement  of  Power I2mo,  3  oo 

Rope  Driving i2mo,  2  oo 

Gill's  Gas  and  Fuel  Analysis  for  Engineers i2mo,  i  25 

Hall's  Car  Lubrication. -      i  zmo,  i  oo 

Button's  The  Gas  Engine 8vo,  5  oa 

Jones's  Machine  Design: 

Part   I.— Kinematics  of  Machinery      8vo.  i  50 

Part  II. — Form,  Strength,  and  Proportions  of  Parts 8vo,  3  oo 

Kent's  Mechanical  Engineer's  Pocket-book i6mo,    morocco,  5  oo 

Kerr's  Power  and  Power  Transmission 8vo,  2  oo 

MacCord's  Kinematics;  or,  Practical  Mechanism 8vo,  5  oo 

Mechanical  Drawing 4to,  4  oo 

Velocity  Diagrams 8vo,  i  50 

11 


Mahan's  Industrial  Drawing.    (Thompson.) 8vo,  3  50 

Poole's  Calorific  Power  of  Fuels 8vo,  3  oo 

Reid's  Course  in  Mechanical  Drawing Svo.  2  oo 

Text-book  of  Mechanical  Drawing  and  Elementary  Machine  Design.  .8vo,  3  oo 

Richards's  Compressed  Air izmo,  i  50 

Robinson's  Principles  of  Mechanism 8vo,  3  oo 

Smith's  Press-working  of  Metals   Svo,  3  oo 

Thurston's  Treatise  on    Friction   and    Lost  Work   in    Machinery   and   Mul 

Work   .              .  8vo,  3   oo 

Animal  as  a  Machine  and  Prime  Motor,  and  the  Laws  of  Energetics.  1 2mo,  i  oo 

Warren's  Elements  of  Machine  Construction  and  Drawing Svo,  750 

Weisbach's  Kinematics  and  the  Power  of  Transmission.      Herrmann- 
Klein.).  .              .  .                   8vo,  5  oo 

Machinery  of  Transmission  and  Governors.     (Herrmann — Klein.).  .Svo,  5  oo 

Hydraul-cs  and  Hydraulic  Motors.     (Du  Bois.) 8vo,  5  oo 

Wolff's  Windmill  as  a  Prime  Mover 8vo,  3  oo 

Wood's  Turbines '. . . : 8vo,  2  50 


MATERIALS  OF  ENGINEERING. 

Bovey's  Strength  of 'Materials  and  Theory  of  Structures 8vo,  7  50 

Burr's  Elasticity  and  Resistance  of  the  Materials  of  Engineering.     6th  Edition, 

Reset 8vo .  7  50 

Church's  Mechanics  of  Engineering Svo,  6  oo 

Johnson'"  Materials  of  Construction Large  8vo,  6  oo 

Keep's  Cast  Iron 8vo,  2  50 

Lanza's  Applied  Mechanics 8vo,  7  50 

Martens's  Handbook  on  Testing  Materials.     (Henning.) 8vo,  7  So 

Merriman's  Text-book  on  the  Mechanics  of  Materials 8vo,  4  oo 

Strength  of  Materals I2mo,  i  oo 

Metcalf's  SteeL     A  Manual  for  Steel-users I2mo  2  oo 

Smith's  Wire :   Its  Use  and  Manufacture Small  410,  3  oo 

Materials  of  Machines I2mo  i  oo 

Thurston's  Materials  of  Engineering 3  vols  ,  Svo,  8  oo 

Part    H.— Iron  and  Steel Svo,  3  50 

Part  IH. — A  Treatise  on  Brasses,  Bronzes,  and  Other  Alloys  and  their 

Constituents Svo  2  50 

Text-book  of  the  Materials  of  Construction Svo,  5  oo 

Wood's  Treatise  on  the  Resistance  of  Materials  and  an  Appendix  on  the 

Preservation  of  Timber Svo,  2  oo 

Elements  of  Analytical  Mechanics Svo,  3  oo 

Wood's  Rustless  Coatings.     (Shortly.) 


STEAM-ENGINES  AND  BOILERS. 

Carnot's  Reflections  on  the  Motive  Power  of  Heat.     (Thurston.) i2mo,  l   50 

Dawson's  "Engineering"  and  Electric  Traction  Pocket-book.  .  i6mo,  mor.,  5  oo 

Ford's  Boiler  Making  for  Boiler  Makers i8mo,  i  oo 

Goss's  Locomotive  Sparks Svo,  2  oo 

Hemt-nway's  Indicator  Practice  and  Steam-engine  Economy i2mo,  2  oo 

Hutton'<?  Mechanical  Engineering  of  Power  Plants Svo,  5  oo 

Heat  and  Heat-engines 8yo.  5  oo 

Kent's  Steam-bo'ler  Economy Svo,  4  oo 

Kneass's  Practice  and  Theory  of  the  Injector Svo  i  50 

MacCord's  Slide-valves Svo,  2  oo 

Meyer's  Modern  Locomotive  Construction 4to.  io  oo 

12 


Peabody's  Manua,  of  the  Steam-engine  Indicator 12 mo,    i  50 

Tables  of  the  Properties  of  Saturated  Steam  and  Other  Vapors 8vo,    i  oo 

Thermodynamics  of  the  Steam-engine  and  Other  Heat-engines 8vo,    5  oo 

Valve-gears  for  Steam-engines    8vo,    2  50 

Peabody  and  Miller's  Steam-boilers         8vo,    4  oo 

Pray's  Twenty  Years  with  the  Indicator  Large  8vo,    2  50 

Pupln's  Thermodynamics  of  Reversible  Cycles  in  Gases  and  Saturated  Vapors. 

(Osterberg.) I2mo.   i  25 

Reagan's  Locomotives  :  Simple,  Compound,  and  Electric I2mo,  2  50 

Rontgen's  Principles  of  Thermodynamics.     (Du  Bois.) 8vo,    5  oo 

Sinclair's  Locomotive  Engine  Running  and  Management i2mo,    2  oo 

Smart's  Handbook  of  Engineering  Laboratory  Practice i2mo,    2  50 

Snow's  Steam-boiler  Practice * 8vo,    3  oo 

Spangler's  Valve-gears 8vo,    2  50 

Notes  on  Thermodynamics   i2mo,    i  oo 

Spangler,  Greene,  and  Marshall's  Elements  of  Steam-engineering. 8vo,    3  oo 

Thurston's  Handy  Tables 8vo.    i    50 

Manual  of  the  Steam-engine 2  vols.   8vo,  10  oo 

Part  I. — History.  Strucruce,  and  Theory 8vo,    6  oo 

Part  H. — Design,  Construction,  and  Operation 8vo,    6  oo 

Handbook  of  Engine  and  Boiler  Trials,  and  the  Use  of  the  Indicator  and 

the  Prony  Brake 8vo,     5  oo 

Stationary  Steant-engines 8vo,    2  50 

Steam-boiler  Explosions  in  Theory  and  in  Practice    12  mo      i   50 

Manual  of  Steam-boiler?  ,  Their  Designs,  Construction,  and  Operation  .  8vo,    5  oo 

Weisbach's  Heat,  Steam,  a    I  Steam-engines.     (Du  Bois  ) 8vo,    5  oo 

Whitham's  Steam-engine  I  ^sign 8vo,    5  oo 

Wilson's  Treatise  on  Steam-boilers.     (Flather.) i6mo,    250 

Wood's  Thermodynamics.  Heat  Motors,  and  Refrigerating  Machines.  .  .  .8vo.    4  oo 


MECHANICS    AND  MACHINERY. 

Barr's  Kinematics  ot  machinery 8vo,  2  50 

Bovey's  Strength  of  Materials  and  Theory  of  Structures 8vo,  7  50 

Chase's  The  Art  of  Pattern-making i2mo,  2  50 

Chordal. — Extracts  from  Letters    I2mo,  2  oo 

Church's  Mechanics  of  Engineering 8vo,  6  oo 

Notes  and  Examples  in  Mechanics 8vo,  2  oo 

Compton's  First  Lessons  in  Metal-working I2mo,  i  50 

Compton  and  De  Groodt's  The  Speed  Lathe i2mo,  i  50 

Cromwell's  Treatise  on  Toothed  Gearing   I2mo,  i  50 

Treatise  on  Belts  and  Pulleys I2mo,  i  50 

Dana's  Text-book  of   Elementary  Mechanics  for  the   Use  of  Colleges  and 

Schools i2mo,  i  50 

Dingey's  Machinery  Pattern  Making i2mo,  2  oo 

Dredge's   Record   of  the   Transportation   Exhibits  Building  of  the   World's 

Columbian  Exposition  of  1803 4to,  half  morocco,  5  oo 

Du  Bois's  Elementary  Principles  of  Mechanics: 

VoL     I. — Kinematics 8vo,  3  50 

Vol     n.— Statics 8vo.  4  oo 

Vol.  HI.— Kinetics 8vo,  3  50 

Mechanics  of  Engineering.     VoL    I Small  4to,  7  50 

Vol.  II Small  4to,  10  oo 

Durley's  Kinematics  of  Machines 8vo,  4  oo 

Fitzgerald's  Boston  Machinist. i6mo,  i  oo 

Flather's  Dynamometers,  and  the  Measurement  of  Power i2mo,  3  oo 

Rope  Driving I2mo,  2  oo 

Goss's  Locomotive  Sparks 8vo,  2  oo 

13 


Hall's  Car  Lubrication i2mo,  i  oo 

Holly's  Art  of  Saw  Filing i8mo  75 

*  Johnson's  Theoretical  Mechanics I2mo,  3  oo 

Statics  by  Graphic  and  Algebraic  Methods 8vo,  2  oo 

Jones's  Machine  Design: 

Part  I. — Kinematics  of  Machinery 8vo,  i  50 

Part  n. — Form,  Strength,  and  Proportions  of  Parts 8vo,  3  oo 

Kerr's  Power  and  Power  Transmission 8vo,  2  oo 

Lanza's  Applied  Mechanics 8vo,  7  50 

MacCord's  Kinematics;  or,  Practical  Mechanism 8*0,  5  oo 

Velocity  Diagrams  8vo.  i  50 

Maurer's  Technical  Mechanics^ 8vo,  4  oo 

Merriman's  Text-book  on  the  Mechanics  of  Materials 8vo,  4  oo 

*  Michie's  Elements  of  Analytical  Mechanics   8vo.  4  oo 

Reagan's  Locomotives:  Simple,  Compound,  and  Electric i2mo,  2  50 

Reid's  Course  in  Mechanical  Drawing 8vo,  2  oo 

Text-book  of  Mechanical  Drawing  and  Elementary  Machine  Design . .  8vo,  3  oo 

Richards's  Compressed  Air i2mo,  i   50 

Robinson's  Principles  of  Mechanism 8vo,  3  oo 

Ryan,  Norris,  and  Hoxie's  Electrical  Machinery 8vo,  2  50 

Sinclair's  Locomotive-engine  Running  and  Management I2mo,  2  oo 

Smith's  Press-working  of  Metals t 8vo,  3  oo 

Materials  of  Machines. i2mo,  i  oo 

Spangler,  Greene,  and  Marshall's  Elements  of  Steam-engineering 8vo,  3  oo 

Thurston's  Treatise  on  Friction  and  Lost  Work  in  Machinery  and  Mill 

Work 8vo,  3  oo 

Animal  as  a  Machine  and  Prime  Motor,  and  the  Laws  of  Energetics.  12  mo,  i  oo 

Warren's  Elements  of  Machine  Construction  and  Drawing 8vo,  7  50 

Weisbach's    Kinematics    and    the   Power  of    Transmission.     (Herrmann — 

Klein.) 8vo,  5  oo 

Machinery  of  Transmission  and  Governors.     (Herrmann — Klein.). 8vo,  5  oo 

Wood's  Elements  of  Analytical  Mechanics 8vo,  3  oo 

Principles  of  Elementary  Mechanics i2mo,  i  25 

Turbines 8vo,  2  50 

The  World's  Columbian  Exposition  of  1893 4to,  i  oo 

METALLURGY. 

Egleston's  Metallurgy  of  Silver,  Gold,  and  Mercury: 

Vol.   I. — Silver 8vo,  7  50 

.  Vol.   H.— Gold  and  Mercury 8vo,  7  So 

**  Iles's  Lead-smelting.     (Postage  9  cents  additional.) i2mo,  2  50 

Keep's  Cast  Iron 8vo,  2  50 

Kunhardt's  Practice  of  Ore  Dressing  in  Europe 8vo,  i   50 

Le  Chatelier's  High-temperature  Measurements.   (Boudouard — Burgess.) .  i2mo,  3  oo 

Metcalf' s  Steel.     A  Manual  for  Steel-users i2mo,  2  oo 

Smith's  Materials  of  Machines I2mo,  i  oo 

Thurston's  Materials  of  Engineering.     In  Three  Parts 8vo,  8  oo 

Part  II. — Iron  and  Steel 8vo,  3  50 

Part  III. — A  Treatise  on  Brasses,  Bronzes,  and  Other  Alloys  and   their 

Constituents 8vo,  2  50 

Ulke's  Modern  Electrolytic  Copper  Refining 8vo,  3  oo 

MINERALOGY. 

Barringer's  Description  of  Minerals  of  Commercial  Value.     Oblong,  morocco,  2  50 

Boyd's  Resources  of  Southwest  Virginia 8vo,  3  oo 

Map  of  Southwest  Virginia Pocket-book  form,  2  oo 

14 


Brush's  Manual  of  Determinative  Mineralogy.     (Penfield.) 8vo,  4  oo 

Chester's  Catalogue  of  Minerals 8vo,  paper,  i  oo 

Cloth,  i  25 

Dictionary  of  the  Names  of  Minerals 8vo,  3  50 

Dana's  System  of  Mineralogy Large  8vo,  half  leather,  12  30 

First  Appendix  to  Dana's  New  "System  of  Mineralogy." Large  8vo,  i  bo 

Text-book  of  Mineralogy 8vo,  4  oo 

Minerals  and  How  to  Study  Them. . . : zarno,  i  50 

Catalogue  of  American  Localities  of  Minerals Large  8vo,  i  oo 

Manual  of  Mineralogy  and  Petrography i2mo,  2  oo 

Bakfe's  Mineral  Tables.     (Shortly.) 

Egleston's  Catalogue  of  Minerals  and  Synonyms 8vo,  2  50 

Hussak's  The  Determination  of  Rock-forming  Minerals.     (Smith.)  Small  8vo,  2  oo 

Merrill's  Non-Metallic  Minerals.     (Shortly.) 

*  Penfield's  Notes  on  Determinative  Mineralogy  and  Record  of  Mineral  Tests. 

8vo,  paper,  o  50 
Rosenbusch's   Microscopical  Physiography   of  the   Rock-making   Minerals. 

(Iddings.) 8vo,  5  oo 

*  Tillman's  Text-book  of  Important  Minerals  and  Docks 8vo,  2  oo 

Wilnams's  Manual  of  Lithology : 8vo,  3  oo 


MINING. 

Beard's  Ventilation  of  Mines i2mo,  2  50 

Boyd's  Resources  of  Southwest  Virginia 8vo,  3  oo 

Map  of  Southwest  Virginia Pocket-book  form,  2  oo 

*  Drinker's  Tunneling,  Explosive  Compounds,  and  Rock  Drills. 

4to,  half  morocco,  25  oo 

Eissler's  Modern  High  Explosives 8vo,  4  oo 

Fowler's  Sewage  Works  Analyses I2mo,  2  oo 

Goodyear 's  Coal-mines  of  the  Western  Coast  of  the  United  States i2mo,  2  50 

Ihlseng's  Manual  of  Mining 8vo,  4  oo 

**  Iles's  Lead-smelting.     (Postage  gc.  additionaL) i2mo,  2  50 

Kunhardt's  Practice  of  Ore  Dressing  in  Europe 8vo,  i  50 

O'Driscoll's  Notes  on  the  Treatment  of  Gold  Ores 8vo,  2  oo 

*  Walke's  Lectures  on  Explosives 8vo,  4  oo 

Wilson's  Cyanide  Processes i2mo,  i  50 

Chlorination  Process I2mo,  i  50 

Hydraulic  and  Placer  Mining i2mo,  2  oo 

Treatise  on  Practical  and  Theoretical  Mine  Ventilation i2mo  i  25 


SANITARY  SCIENCE. 

Copeland's  Manual  of  Bacteriology.     (In  preparation.) 

FolwelTs  Sewerage.     (Designing,  Construction  and  Maintenance.; 8vo,  3  oo 

Water-supply  Engineering 8vo,  4  oo 

Fuertes's  Water  and  Public  Health xamo ,  i  50 

Water-filtration   Works X2mo,  2  50 

Gerhard's  Guide  to  Sanitary  House-inspection i6mo,  i  oo 

Goodrich's  Economical  Disposal  of  Town's  Refuse Demy  8vo,  3  50 

Hazen's  Filtration  of  Public  Water-supplies 8vo,  3  oo 

Kiersted's  Sewage  Disposal X2mo,  i  25 

Loach's  The  Inspection  and  Analysis  of  Food  with  Special  Reference  to  State 

Control.     (In  preparation.) 

Mason's   Water-supply.     (Considered   Principally   from   a   Sanitary   Stand- 
point.)   3d  Edition,  Rewritten 8vo,  4  oo 

Examination  of  Water.     (Chemical  and  Bacteriological.) 12 mo,  i  25 

15 


Merriman's  Elements  of  Sanitary  Engineering   8vo,  2  oo 

Nichols's  Water-supply.     (Considered  Mainly  from  a  Chemical  and  Sanitary 

Standpoint.)     (1883.) 8vo,  50 

Ogden's  Sewer  Design i2mo,  oo 

*  Price's  Handbook  on  Sanitation i2mo,  50 

Richards's  Cost  of  Food.     A  Study  in  Dietaries   i2mo,  oo 

Cost  of  Living  as  Modified  by  Sanitary  Science i2mo,  oo 

Richards  and  Woodman's  Air,  Water,  and  Food  from  a  Sanitary  Stand- 
point   8vo,  2  oo 

*  Richards  and  Williams's  The  Dietary  Computer 8vo,  i  50 

Ri deal's  Sewage  and  Bacterial  Purification  of  Sewage 8vo,  3  50 

Turneaure  and  Russell's  Public  Water-supplies 8vo,  5  oo 

Whipple's  Microscopy  of  Drinking-water 8vo,  3  50 

Woodhull's  Notes  and  Military  Hygiene i6mo,  i  50 

MISCELLANEOUS. 

Barker's  Deep-sea  Soundings 8vo,  2  oo 

Emmons's  Geological  Guide-book  of  the  Rocky  Mountain  Excursion  of  the 

International  Congress  of  Geologists Large  8vo,  50 

Ferrel's  Popular  Treatise  on  the  Winds 8vo,  oo 

Haines's  American  Railway  Management i2mo,  50 

Mott's  Composition,  Digestibility ,  and  Nutritive  Value  of  Food.   Mounted  chart.  25 

Fallacy  of  the  Present  Theory  of  Sound i6mo,  oo 

Ricketts's  History  of  Rensselaer  Polytechnic  Institute,  1824-1894.  Small  8vo,  3  oo 

Rotherham's  Emphasized  New  Testament Large  8vo,  2  oo 

Steel's  Treatise  on  the  Diseases  of  the  Dog 8vo,  3  50 

Totten's  Important  Question  in  Metrology 8vo,  2  50 

The  World's  Columbian  Exposition  ot  1893 4to,  i  oo 

Worcester  and  Atkinson.     Small  Hospitals,  Establishment  and  Maintenance, 
and  Suggestions  for  Hospital  Architecture,  with  Plans  for  a  Small 

Hospital i2mo,  i   25 

HEBREW  AND  CHALDEE  TEXT-BOOKS. 

Green's  Grammar  of  the  Hebrew  Language 8vo,  3  oo 

Elementary  Hebrew  Grammar i  amo,  i   25 

Hebrew  Chrestomathy   8vo,  2  oo 

Gesenius's  Hebrew  and  Chaldee  Lexicon  to   the  Old  Testament  Scriptures. 

(Tregelles.) . . ; Small  4to,  half  morocco,  5 j oo 

Letteris's  Hebrew  Bible 8vo,  2  25 

16 


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